Genetic polymorphisms associated with coronary stenosis, methods of detection and uses thereof

ABSTRACT

The present invention is based on the discovery of genetic polymorphisms that are associated with coronary stenosis. In particular, the present invention relates to nucleic acid molecules containing the polymorphisms, variant proteins encoded by such nucleic acid molecules, reagents for detecting the polymorphic nucleic acid molecules and proteins, and methods of using the nucleic acid and proteins as well as methods of using reagents for their detection.

FIELD OF THE INVENTION

The present invention is in the field of stenosis diagnosis and therapy.In particular, the present invention relates to specific singlenucleotide polymorphisms (SNPs) in the human genome, and theirassociation with stenosis and related pathologies. Based on differencesin allele frequencies in the stenosis patient population relative tonormal individuals, the naturally-occurring SNPs disclosed herein can beused as targets for the design of diagnostic reagents and thedevelopment of therapeutic agents, as well as for disease associationand linkage analysis. In particular, the SNPs of the present inventionare useful for identifying an individual who is at an increased ordecreased risk of developing stenosis and for early detection of thedisease, for providing clinically important information for theprevention and/or treatment of stenosis, and for screening and selectingtherapeutic agents. The SNPs disclosed herein are also useful for humanidentification applications. Methods, assays, kits, and reagents fordetecting the presence of these polymorphisms and their encoded productsare provided.

BACKGROUND OF THE INVENTION

Stenosis

Coronary stenosis is the narrowing of coronary arteries by obstructiveatherosclerotic plaques. The coronary arteries supply oxygenated bloodflow to the myocardium. Although mild and moderate coronary stenosis donot impede resting coronary flow, stenosis >30-45% starts to restrictmaximal coronary flow. Severe coronary stenosis (>70% reduction inluminal diameter) causes stable angina (ischemic chest pain uponexertion). Significant stenosis contributes, along with plaque ruptureand thrombus formation, coronary spasm, or inflammation/infection, tounstable angina as well as myocardial infarction. Together witharrhythmia, coronary stenosis is a major factor of sudden cardiacdeaths, as evidenced by its presence in two or more major coronaryarteries in 90% of adult sudden cardiac death victims.

Coronary stenosis is a prevalent disease. Each year in the UnitedStates, 440,000 new cases of stable angina and 150,000 new cases ofunstable angina occur. This year, an estimated 1.1 million Americanswill have a new or recurrent heart attack. These incidences result inover six million individuals in the U.S. living with stable or unstableangina pectoris, a debilitating condition, and over seven millionindividuals in the U.S. living with a history of myocardial infarction.Coronary stenosis is frequently a deadly disease. It is a majorunderlying cause of coronary heart disease (CHD), which is the singlelargest cause of death in the U.S. Over half a million coronary deaths,including 250,000 sudden cardiac deaths, occur each year in U.S.Coronary stenosis is also a costly disease. It is the major reason for1.2 million cardiac catheterizations, 0.4 million angioplasties, and 0.6million bypass surgeries, contributing to the estimated 110 billiondollar total costs of CHD in the U.S. for the year 2002. Still, thesestatistics underestimate the true prevalence of the disease sincecoronary stenosis often remains clinically asymptomatic for decades, andonly becomes symptomatic when the disease has progressed to a severe,and sometime fatal, state.

There is therefore an unmet need in early diagnosis and prognosis ofasymptomatic coronary stenosis. This need is particularly significantgiven that early diagnosis or prognosis results can significantlyinfluence the course of disease by influencing treatment choices (forexample, those with genetic risks can be treated to modify risk factorssuch as hypertension, diabetes, inactivity, dyslipidemia, etc.),thresholds (e.g., lipid levels used to trigger the use of lipid-loweringdrugs), and goals (e.g., target blood pressure or lipid levels), andpossibly enhance compliance.

Diagnosis of coronary stenosis currently starts by assessing if the riskprofiles (e.g., hypertension, dyslipidemia, family history, diabetes,etc.) and symptoms (e.g., angina) of patients are consistent withcoronary heart disease, followed most commonly by resting and exerciseEKGs. However, risk assessments and EKGs are imperfect diagnostic testsfor stenosis since they can be both insensitive (giving false negatives)and non-specific (giving false positives). Coronary arteriography is thedefinitive test for assessing the severity of coronary stenosis,however, it is not very sensitive in early detection of mild stenosis.It is also an invasive procedure with a small risk of death due to thecatheterization procedure and the contrast dye. Because of this risk, itis typically only used at a time when coronary stenosis is consideredlikely from symptoms or other tests, which is hardly an ideal time tostart intervention.

Coronary stenosis risk is presumed to have a strong genetic component.It is well known that several major risk factors of coronary disease areheritable, e.g. serum lipid levels (Perusse L. et. al., ArteriosclerThromb Vasc Biol (1997): 17(11) 3263-9) and obesity (Rice T. et. al.,Int J Obes Relat Metab Disord (1997):21(11) 1024-31). Indeed, severalknown genetic defects are individually sufficient to cause elevatedserum LDL-cholesterol (e.g., familial hypercholesterolemia) leading topremature coronary disease (Goldstein and Brown, Science 292 (2001):1310-12). In addition, linkage studies in humans have replicated thefindings of the link of several chromosomal regions (quantitative traitloci) to coronary heart disease and related diseases and risk factors(Pajukanta P. et. al., Am J Hum Genet 67 (2000):1481-93, Francke S. et.al., Human Molecular Genetics (2001): 10 (24) 2751-65). Finally, afamily history of premature coronary disease is a significant factor inthe risk assessment and diagnosis of coronary disease (Braunwald E.,Zipes D. and Libby P., Heart Disease, 6^(th) ed. W. B. Saunders Company,2001, 28).

Although many risk factors for coronary stenosis have been identified,including age, diabetes, hypertension, high serum cholesterol, smoking,etc., and genetic factors play significant roles in several of theserisk factors, significant genetic risk factors are likely to exist whichhave not been identified to date. In addition to the anecdotal coronarydisease patients that exhibit few traditional risk factors, a study ofmultiple existing risk factors showed that only half of the“population-attributable risk” was attributable to known risk factors(Change M. et. al., J Clin Epidemiol (2001) 54 (6) 634-44). Therefore,the presently known risk factors are inadequate for predicting coronarystenosis risk in individuals. Given the magnitude of the disease, thereis an urgent need for genetic markers that are predictive of coronarystenosis risk. Such genetic markers could increase the prognosticability of existing risk assessment methods and complement currentdiagnostic methods such as exercise EKG, especially in early detectionof disease when intervention is most effective and should ideally start.

SNPs

The genomes of all organisms undergo spontaneous mutation in the courseof their continuing evolution, generating variant forms of progenitorgenetic sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). Avariant form may confer an evolutionary advantage or disadvantagerelative to a progenitor form or may be neutral. In some instances, avariant form confers an evolutionary advantage to the species and iseventually incorporated into the DNA of many or most members of thespecies and effectively becomes the progenitor form. Additionally, theeffects of a variant form may be both beneficial and detrimental,depending on the circumstances. For example, a heterozygous sickle cellmutation confers resistance to malaria, but a homozygous sickle cellmutation is usually lethal. In many cases, both progenitor and variantforms survive and co-exist in a species population. The coexistence ofmultiple forms of a genetic sequence gives rise to geneticpolymorphisms, including SNPs.

Approximately 90% of all polymorphisms in the human genome are SNPs.SNPs are single base positions in DNA at which different alleles, oralternative nucleotides, exist in a population. The SNP position(interchangeably referred to herein as SNP, SNP site, or SNP locus) isusually preceded by and followed by highly conserved sequences of theallele (e.g., sequences that vary in less than 1/100 or 1/1000 membersof the populations). An individual may be homozygous or heterozygous foran allele at each SNP position. A SNP can, in some instances, bereferred to as a “cSNP” to denote that the nucleotide sequencecontaining the SNP is an amino acid coding sequence.

A SNP may arise from a substitution of one nucleotide for another at thepolymorphic site. Substitutions can be transitions or transversions. Atransition is the replacement of one purine nucleotide by another purinenucleotide, or one pyrimidine by another pyrimidine. A transversion isthe replacement of a purine by a pyrimidine, or vice versa. A SNP mayalso be a single base insertion or deletion variant referred to as an“indel” (Weber et al., “Human diallelic insertion/deletionpolymorphisms”, Am J Hum Genet 2002 October; 71(4):854-62).

A synonymous codon change, or silent mutation/SNP (terms such as “SNP”,“polymorphism”, “mutation”, “mutant”, “variation”, and “variant” areused herein interchangeably), is one that does not result in a change ofamino acid due to the degeneracy of the genetic code. A substitutionthat changes a codon coding for one amino acid to a codon coding for adifferent amino acid (i.e., a non-synonymous codon change) is referredto as a missense mutation. A nonsense mutation results in a type ofnon-synonymous codon change in which a stop codon is formed, therebyleading to premature termination of a polypeptide chain and a truncatedprotein. A read-through mutation is another type of non-synonymous codonchange that causes the destruction of a stop codon, thereby resulting inan extended polypeptide product. While SNPs can be bi-, tri-, ortetra-allelic, the vast majority of the SNPs are bi-allelic, and arethus often referred to as “bi-allelic markers”, or “di-allelic markers”.

As used herein, references to SNPs and SNP genotypes include individualSNPs and/or haplotypes, which are groups of SNPs that are generallyinherited together. Haplotypes can have stronger correlations withdiseases or other phenotypic effects compared with individual SNPs, andtherefore may provide increased diagnostic accuracy in some cases(Stephens et al. Science 293, 489-493, 20 July 2001).

Causative SNPs are those SNPs that produce alterations in geneexpression or in the expression, structure, and/or function of a geneproduct, and therefore are most predictive of a possible clinicalphenotype. One such class includes SNPs falling within regions of genesencoding a polypeptide product, i.e. cSNPs. These SNPs may result in analteration of the amino acid sequence of the polypeptide product (i.e.,non-synonymous codon changes) and give rise to the expression of adefective or other variant protein. Furthermore, in the case of nonsensemutations, a SNP may lead to premature termination of a polypeptideproduct. Such variant products can result in a pathological condition,e.g., genetic disease. Examples of genes in which a SNP within a codingsequence causes a genetic disease include sickle cell anemia and cysticfibrosis.

Causative SNPs do not necessarily have to occur in coding regions;causative SNPs can occur in, for example, any genetic region that canultimately affect the expression, structure, and/or activity of theprotein encoded by a nucleic acid. Such genetic regions include, forexample, those involved in transcription, such as SNPs in transcriptionfactor binding domains, SNPs in promoter regions, in areas involved intranscript processing, such as SNPs at intron-exon boundaries that maycause defective splicing, or SNPs in mRNA processing signal sequencessuch as polyadenylation signal regions. Some SNPs that are not causativeSNPs nevertheless are in close association with, and therefore segregatewith, a disease-causing sequence. In this situation, the presence of aSNP correlates with the presence of, or predisposition to, or anincreased risk in developing the disease. These SNPs, although notcausative, are nonetheless also useful for diagnostics, diseasepredisposition screening, and other uses.

An association study of a SNP and a specific disorder involvesdetermining the presence or frequency of the SNP allele in biologicalsamples from individuals with the disorder of interest, such asstenosis, and comparing the information to that of controls (i.e.,individuals who do not have the disorder; controls may be also referredto as “healthy” or “normal” individuals) who are preferably of similarage and race. The appropriate selection of patients and controls isimportant to the success of SNP association studies. Therefore, a poolof individuals with well-characterized phenotypes is extremelydesirable.

A SNP may be screened in diseased tissue samples or any biologicalsample obtained from a diseased individual, and compared to controlsamples, and selected for its increased (or decreased) occurrence in aspecific pathological condition, such as pathologies related tostenosis. Once a statistically significant association is establishedbetween one or more SNP(s) and a pathological condition (or otherphenotype) of interest, then the region around the SNP can optionally bethoroughly screened to identify the causative genetic locus/sequence(s)(e.g., causative SNP/mutation, gene, regulatory, region, etc.) thatinfluences the pathological condition or phenotype. Association studiesmay be conducted within the general population and are not limited tostudies performed on related individuals in affected families (linkagestudies).

Clinical trials have shown that patient response to treatment withpharmaceuticals is often heterogeneous. There is a continuing need toimprove pharmaceutical agent design and therapy. In that regard, SNPscan be used to identify patients most suited to therapy with particularpharmaceutical agents (this is often termed “pharmacogenomics”).Similarly, SNPs can be used to exclude patients from certain treatmentdue to the patient's increased likelihood of developing toxic sideeffects or their likelihood of not responding to the treatment.Pharmacogenomics can also be used in pharmaceutical research to assistthe drug development and selection process. (Linder et al. (1997),Clinical Chemistry, 43, 254; Marshall (1997), Nature Biotechnology, 15,1249; International Patent Application WO 97/40462, Spectra Biomedical;and Schafer et al. (1998), Nature Biotechnology, 16, 3).

SUMMARY OF THE INVENTION

The present invention relates to the identification of novel SNPs,unique combinations of such SNPs, and haplotypes of SNPs that areassociated with stenosis and related pathologies. The polymorphismsdisclosed herein are directly useful as targets for the design ofdiagnostic reagents and the development of therapeutic agents for use inthe diagnosis and treatment of stenosis and related pathologies.

Based on the identification of SNPs associated with stenosis, thepresent invention also provides methods of detecting these variants aswell as the design and preparation of detection reagents needed toaccomplish this task. The invention specifically provides novel SNPs ingenetic sequences involved in stenosis, variant proteins encoded bynucleic acid molecules containing such SNPs, antibodies to the encodedvariant proteins, computer-based and data storage systems containing thenovel SNP information, methods of detecting these SNPs in a test sample,methods of identifying individuals who have an altered (i.e., increasedor decreased) risk of developing stenosis based on the presence of a SNPdisclosed herein or its encoded product, methods of identifyingindividuals who are more or less likely to respond to a treatment,methods of screening for compounds useful in the treatment of a disorderassociated with a variant gene/protein, compounds identified by thesemethods, methods of treating disorders mediated by a variantgene/protein, and methods of using the novel SNPs of the presentinvention for human identification.

In Tables 1-2, the present invention provides gene information,transcript sequences (SEQ ID NOS:1-284), encoded amino acid sequences(SEQ ID NOS:285-568), genomic sequences (SEQ ID NOS:5608-5780),transcript-based context sequences (SEQ ID NOS:569-5607) andgenomic-based context sequences (SEQ ID NOS:5781-26,130) that containthe SNPs of the present invention, and extensive SNP information thatincludes observed alleles, allele frequencies, populations/ethnic groupsin which alleles have been observed, information about the type of SNPand corresponding functional effect, and, for cSNPs, information aboutthe encoded polypeptide product. The transcript sequences (SEQ IDNOS:1-284), amino acid sequences (SEQ ID NOS:285-568), genomic sequences(SEQ ID NOS:5608-5780), transcript-based SNP context sequences (SEQ IDNOS: 569-5607), and genomic-based SNP context sequences (SEQ IDNOS:5781-26,130) are also provided in the Sequence Listing.

In a specific embodiment of the present invention, naturally-occurringSNPs in the human genome are provided. These SNPs are associated withstenosis such that they can have a variety of uses in the diagnosisand/or treatment of stenosis. One aspect of the present inventionrelates to an isolated nucleic acid molecule comprising a nucleotidesequence in which at least one nucleotide is a SNP disclosed in Tables 3and/or 4. In an alternative embodiment, a nucleic acid of the inventionis an amplified polynucleotide, which is produced by amplification of aSNP-containing nucleic acid template. In another embodiment, theinvention provides for a variant protein which is encoded by a nucleicacid molecule containing a SNP disclosed herein.

In yet another embodiment of the invention, a reagent for detecting aSNP in the context of its naturally-occurring flanking nucleotidesequences (which can be, e.g., either DNA or mRNA) is provided. Inparticular, such a reagent may be in the form of, for example, ahybridization probe or an amplification primer that is useful in thespecific detection of a SNP of interest. In an alternative embodiment, aprotein detection reagent is used to detect a variant protein which isencoded by a nucleic acid molecule containing a SNP disclosed herein. Apreferred embodiment of a protein detection reagent is an antibody or anantigen-reactive antibody fragment.

Also provided in the invention are kits comprising SNP detectionreagents, and methods for detecting the SNPs disclosed herein byemploying detection reagents. In a specific embodiment, the presentinvention provides for a method of identifying an individual having anincreased or decreased risk of developing stenosis by detecting thepresence or absence of a SNP allele disclosed herein. In anotherembodiment, a method for diagnosis of stenosis by detecting the presenceor absence of a SNP allele disclosed herein is provided.

The nucleic acid molecules of the invention can be inserted in anexpression vector, such as to produce a variant protein in a host cell.Thus, the present invention also provides for a vector comprising aSNP-containing nucleic acid molecule, genetically-engineered host cellscontaining the vector, and methods for expressing a recombinant variantprotein using such host cells. In another specific embodiment, the hostcells, SNP-containing nucleic acid molecules, and/or variant proteinscan be used as targets in a method for screening and identifyingtherapeutic agents or pharmaceutical compounds useful in the treatmentof stenosis.

An aspect of this invention is a method for treating stenosis in a humansubject wherein said human subject harbors a gene, transcript, and/orencoded protein identified in Tables 1-2, which method comprisesadministering to said human subject a therapeutically orprophylactically effective amount of one or more agents counteractingthe effects of the disease, such as by inhibiting (or stimulating) theactivity of the gene, transcript, and/or encoded protein identified inTables 1-2.

Another aspect of this invention is a method for identifying an agentuseful in therapeutically or prophylactically treating stenosis in ahuman subject wherein said human subject harbors a gene, transcript,and/or encoded protein identified in Tables 1-2, which method comprisescontacting the gene, transcript, or encoded protein with a candidateagent under conditions suitable to allow formation of a binding complexbetween the gene, transcript, or encoded protein and the candidate agentand detecting the formation of the binding complex, wherein the presenceof the complex identifies said agent.

Another aspect of this invention is a method for treating stenosis in ahuman subject, which method comprises:

(i) determining that said human subject harbors a gene, transcript,and/or encoded protein identified in Tables 1-2, and

(ii) administering to said subject a therapeutically or prophylacticallyeffective amount of one or more agents counteracting the effects of thedisease.

Many other uses and advantages of the present invention will be apparentto those skilled in the art upon review of the detailed description ofthe preferred embodiments herein. Solely for clarity of discussion, theinvention is described in the sections below by way of non-limitingexamples.

Description of the Files Contained on the CD-R Named CL001500CDR

The CD-R named CL001500CDR contains the following five text (ASCII)files:

1) File SEQLIST_(—)1500.txt provides the Sequence Listing. The SequenceListing provides the transcript sequences (SEQ ID NOS:1-284) and proteinsequences (SEQ ID NOS:285-568) as shown in Table 1, and genomicsequences (SEQ ID NOS:5608-5780) as shown in Table 2, for eachstenosis-associated gene that contains one or more SNPs of the presentinvention. Also provided in the Sequence Listing are context sequencesflanking each SNP, including both transcript-based context sequences asshown in Table 1 (SEQ ID NOS:569-5607) and genomic-based contextsequences as shown in Table 2 (SEQ ID NOS:5781-26,130). The contextsequences generally provide 100 bp upstream (5′) and 100 bp downstream(3′) of each SNP, with the SNP in the middle of the context sequence,for a total of 200 bp of context sequence surrounding each SNP. FileSEQLIST_(—)1500.txt is 23,557 KB in size.

2) File TABLE1_(—)1500.txt provides Table 1. File TABLE1_(—)1500.txt is4,270 KB in size.

3) File TABLE2_(—)1500.txt provides Table 2. File TABLE2_(—)1500.txt is19,508 KB in size.

4) File TABLE3_(—)1500.txt provides Table 3. File TABLE3_(—)1500.txt is34 KB in size.

5) File TABLE4_(—)1500.txt provides Table 4. File TABLE4_(—)1500.txt is59 KB in size.

The material contained on the CD-R labeled CL001500CDR is herebyincorporated by reference pursuant to 37 CFR 1.77(b)(4).

Description of Table 1 and Table 2

Table 1 and Table 2 (both provided on the CD-R) disclose the SNP andassociated gene/transcript/protein information of the present invention.For each gene, Table 1 and Table 2 each provide a header containinggene/transcript/protein information, followed by a transcript andprotein sequence (in Table 1) or genomic sequence (in Table 2), and thenSNP information regarding each SNP found in that gene/transcript.

NOTE: SNPs may be included in both Table 1 and Table 2; Table 1 presentsthe SNPs relative to their transcript sequences and encoded proteinsequences, whereas Table 2 presents the SNPs relative to their genomicsequences (in some instances Table 2 may also include, after the lastgene sequence, genomic sequences of one or more intergenic regions, aswell as SNP context sequences and other SNP information for any SNPsthat lie within these intergenic regions). SNPs can readily becross-referenced between Tables based on their hCV (or, in someinstances, hDV) identification numbers.

The gene/transcript/protein information includes:

-   -   a gene number (1 through n, where n=the total number of genes in        the Table)    -   a Celera hCG and UID internal identification numbers for the        gene    -   a Celera hCT and UID internal identification numbers for the        transcript (Table 1 only)    -   a public Genbank accession number (e.g., RefSeq NM number) for        the transcript (Table 1 only)    -   a Celera hCP and UID internal identification numbers for the        protein encoded by the hCT transcript (Table 1 only)    -   a public Genbank accession number (e.g., RefSeq NP number) for        the protein (Table 1 only)    -   an art-known gene symbol    -   an art-known gene/protein name    -   Celera genomic axis position (indicating start nucleotide        position-stop nucleotide position)    -   the chromosome number of the chromosome on which the gene is        located    -   an OMIM (Online Mendelian Inheritance in Man; Johns Hopkins        University/NCBI) public reference number for obtaining further        information regarding the medical significance of each gene    -   alternative gene/protein name(s) and/or symbol(s) in the OMIM        entry

NOTE: Due to the presence of alternative splice forms, multipletranscript/protein entries can be provided for a single gene entry inTable 1; i.e., for a single Gene Number, multiple entries may beprovided in series that differ in their transcript/protein informationand sequences.

Following the gene/transcript/protein information is a transcriptsequence and protein sequence (in Table 1), or a genomic sequence (inTable 2), for each gene, as follows:

-   -   transcript sequence (Table 1 only) (corresponding to SEQ ID        NOS:1-284 of the Sequence Listing), with SNPs identified by        their IUB codes (transcript sequences can include 5′ UTR,        protein coding, and 3′ UTR regions). (NOTE: If there are        differences between the nucleotide sequence of the hCT        transcript and the corresponding public transcript sequence        identified by the Genbank accession number, the hCT transcript        sequence (and encoded protein) is provided, unless the public        sequence is a RefSeq transcript sequence identified by an NM        number, in which case the RefSeq NM transcript sequence (and        encoded protein) is provided. However, whether the hCT        transcript or RefSeq NM transcript is used as the transcript        sequence, the disclosed SNPs are represented by their IUB codes        within the transcript.)    -   the encoded protein sequence (Table 1 only) (corresponding to        SEQ ID NOS:285-568 of the Sequence Listing)    -   the genomic sequence of the gene (Table 2 only), including 6 kb        on each side of the gene boundaries (i.e., 6 kb on the 5′ side        of the gene plus 6 kb on the 3′ side of the gene) (corresponding        to SEQ ID NOS:5608-5780 of the Sequence Listing).

After the last gene sequence, Table 2 may include additional genomicsequences of intergenic regions (in such instances, these sequences areidentified as “Intergenic region:” followed by a numericalidentification number), as well as SNP context sequences and other SNPinformation for any SNPs that lie within each intergenic region (andsuch SNPs are identified as “INTERGENIC” for SNP type).

NOTE: The transcript, protein, and transcript-based SNP contextsequences are provided in both Table 1 and in the Sequence Listing. Thegenomic and genomic-based SNP context sequences are provided in bothTable 2 and in the Sequence Listing. SEQ ID NOS are indicated in Table 1for each transcript sequence (SEQ ID NOS:1-284), protein sequence (SEQID NOS:285-568), and transcript-based SNP context sequence (SEQ IDNOS:569-5607), and SEQ ID NOS are indicated in Table 2 for each genomicsequence (SEQ ID NOS:5608-5780), and genomic-based SNP context sequence(SEQ ID NOS:5781-26,130).

The SNP information includes:

-   -   context sequence (taken from the transcript sequence in Table 1,        and taken from the genomic sequence in Table 2) with the SNP        represented by its IUB code, including 100 bp upstream (5′) of        the SNP position plus 100 bp downstream (3′) of the SNP position        (the transcript-based SNP context sequences in Table 1 are        provided in the Sequence Listing as SEQ ID NOS:569-5607; the        genomic-based SNP context sequences in Table 2 are provided in        the Sequence Listing as SEQ ID NOS:5781-26,130).    -   Celera hCV internal identification number for the SNP (in some        instances, an “hDV” number is given instead of an “hCV” number)    -   SNP position [position of the SNP within the given transcript        sequence (Table 1) or within the given genomic sequence (Table        2)]    -   SNP source (may include any combination of one or more of the        following five codes, depending on which internal sequencing        projects and/or public databases the SNP has been observed in:        “Applera”=SNP observed during the re-sequencing of genes and        regulatory regions of 39 individuals, “Celera”=SNP observed        during shotgun sequencing and assembly of the Celera human        genome sequence, “Celera Diagnostics”=SNP observed during        re-sequencing of nucleic acid samples from individuals who have        stenosis or a related pathology, “dbSNP”=SNP observed in the        dbSNP public database, “HGBASE”=SNP observed in the HGBASE        public database, “HGMD”=SNP observed in the Human Gene Mutation        Database (HGMD) public database) (NOTE: multiple “Applera”        source entries for a single SNP indicate that the same SNP was        covered by multiple overlapping amplification products and the        re-sequencing results (e.g., observed allele counts) from each        of these amplification products is being provided)    -   Population/allele/allele count information in the format of        [population1(allele1,count|allele2,count)        population2(allele1,count|allele2,count) total (allele1,total        count|allele2,total count)]. The information in this field        includes populations/ethnic groups in which particular SNP        alleles have been observed (“cau”=Caucasian, “his”=Hispanic,        “chn”=Chinese, and “afr”=African-American, “jpn”=Japanese,        “ind”=Indian, “mex”=Mexican, “ain”=“American Indian,        “cra”=Celera donor, “no_pop”=no population information        available), identified SNP alleles, and observed allele counts        (within each population group and total allele counts), where        available [“−” in the allele field represents a deletion allele        of an insertion/deletion (“indel”) polymorphism (in which case        the corresponding insertion allele, which may be comprised of        one or more nucleotides, is indicated in the allele field on the        opposite side of the “|”); “−” in the count field indicates that        allele count information is not available].

NOTE: For SNPs of “Applera” SNP source, genes/regulatory regions of 39individuals (20 Caucasians and 19 African Americans) were re-sequencedand, since each SNP position is represented by two chromosomes in eachindividual (with the exception of SNPs on X and Y chromosomes in males,for which each SNP position is represented by a single chromosome), upto 78 chromosomes were genotyped for each SNP position. Thus, the sum ofthe African-American (“afr”) allele counts is up to 38, the sum of theCaucasian allele counts (“cau”) is up to 40, and the total sum of allallele counts is up to 78.

(NOTE: semicolons separate population/allele/count informationcorresponding to each indicated SNP source; i.e., if four SNP sourcesare indicated, such as “Celera”, “dbSNP”, “HGBASE”, and “HGMD”, thenpopulation/allele/count information is provided in four groups which areseparated by semicolons and listed in the same order as the listing ofSNP sources, with each population/allele/count information groupcorresponding to the respective SNP source based on order; thus, in thisexample, the first population/allele/count information group wouldcorrespond to the first listed SNP source (Celera) and the thirdpopulation/allele/count information group separated by semicolons wouldcorrespond to the third listed SNP source (HGBASE); ifpopulation/allele/count information is not available for any particularSNP source, then a pair of semicolons is still inserted as aplace-holder in order to maintain correspondence between the list of SNPsources and the corresponding listing of population/allele/countinformation)

-   -   SNP type (e.g., location within gene/transcript and/or predicted        functional effect) [“MIS-SENSE MUTATION”=SNP causes a change in        the encoded amino acid (i.e., a non-synonymous coding SNP);        “SILENT MUTATION”=SNP does not cause a change in the encoded        amino acid (i.e., a synonymous coding SNP); “STOP CODON        MUTATION”=SNP is located in a stop codon; “NONSENSE        MUTATION”=SNP creates or destroys a stop codon; “UTR 5”=SNP is        located in a 5′ UTR of a transcript; “UTR 3”=SNP is located in a        3′ UTR of a transcript; “PUTATIVE UTR 5”=SNP is located in a        putative 5′ UTR; “PUTATIVE UTR 3”=SNP is located in a putative        3′ UTR; “DONOR SPLICE SITE”=SNP is located in a donor splice        site (5′ intron boundary); “ACCEPTOR SPLICE SITE”=SNP is located        in an acceptor splice site (3′ intron boundary); “CODING        REGION”=SNP is located in a protein-coding region of the        transcript; “EXON”=SNP is located in an exon; “INTRON”=SNP is        located in an intron; “hmCS”=SNP is located in a human-mouse        conserved segment; “TFBS”=SNP is located in a transcription        factor binding site; “UNKNOWN”=SNP type is not defined;        “INTERGENIC”=SNP is intergenic, i.e., outside of any gene        boundary]    -   Protein coding information (Table 1 only), where relevant, in        the format of [protein SEQ ID NO:#, amino acid position, (amino        acid-1, codon1) (amino acid-2, codon2)]. The information in this        field includes SEQ ID NO of the encoded protein sequence,        position of the amino acid residue within the protein identified        by the SEQ ID NO that is encoded by the codon containing the        SNP, amino acids (represented by one-letter amino acid codes)        that are encoded by the alternative SNP alleles (in the case of        stop codons, “X” is used for the one-letter amino acid code),        and alternative codons containing the alternative SNP        nucleotides which encode the amino acid residues (thus, for        example, for missense mutation-type SNPs, at least two different        amino acids and at least two different codons are generally        indicated; for silent mutation-type SNPs, one amino acid and at        least two different codons are generally indicated, etc.). In        instances where the SNP is located outside of a protein-coding        region (e.g., in a UTR region), “None” is indicated following        the protein SEQ ID NO.

Description of Table 3 and Table 4

Tables 3 and 4 (both provided on the CD-R) provide a list of a subset ofSNPs from Table 1 (in the case of Table 3) or Table 2 (in the case ofTable 4) for which the SNP source falls into one of the following threecategories: 1) SNPs for which the SNP source is only “Applera” and noneother, 2) SNPs for which the SNP source is only “Celera Diagnostics” andnone other, and 3) SNPs for which the SNP source is both “Applera” and“Celera Diagnostics” but none other.

These SNPs have not been observed in any of the public databases (dbSNP,HGBASE, and HGMD), and were also not observed during shotgun sequencingand assembly of the Celera human genome sequence (i.e., “Celera” SNPsource). Tables 3 and 4 provide the hCV identification number (or hDVidentification number for SNPs having “Celera Diagnostics” SNP source)and the SEQ ID NO of the context sequence for each of these SNPs.

Description of Table 5

Table 5 provides sequences (SEQ ID NOS:26,131-26,415) of primers thathave been synthesized and used in the laboratory to carry outallele-specific PCR reactions in order to assay the SNPs disclosed inTables 6-7 during the course of stenosis association studies.

Table 5 provides the following:

-   -   the column labeled “hCV” provides an hCV identification number        for each SNP site    -   the column labeled “Alleles” designates the two alternative        alleles at the SNP site identified by the hCV identification        number that are targeted by the allele-specific primers (the        allele-specific primers are shown as “Sequence A” and “Sequence        B” in each row)    -   the column labeled “Sequence A (allele-specific primer)”        provides an allele-specific primer that is specific for the        first allele designated in the “Alleles” column    -   the column labeled “Sequence B (allele-specific primer)”        provides an allele-specific primer that is specific for the        second allele designated in the “Alleles” column    -   the column labeled “Sequence C (common primer)” provides a        common primer that is used in conjunction with each of the        allele-specific primers (the “Sequence A” primer and the        “Sequence B” primer) and which hybridizes at a site away from        the SNP position.

All primer sequences are given in the 5′ to 3′ direction.

Each of the alleles designated in the “Alleles” column matches the 3′nucleotide of the allele-specific primer that is specific for thatallele. Thus, the first allele designated in the “Alleles” columnmatches the 3′ nucleotide of the “Sequence A” primer, and the secondallele designated in the “Alleles” column matches the 3′ nucleotide ofthe “Sequence B” primer.

Description of Table 6 and Table 7

Tables 6-7 provide results of statistical analyses for SNPs disclosed inTables 1-5 (SNPs can be cross-referenced between Tables based on theirhCV identification numbers). The statistical results provide support forthe association of these SNPs with coronary stenosis.

NOTE: SNPs can be cross-referenced between Tables 1-7 based on the hCVidentification number of each SNP. However, six of the SNPs that areincluded in Tables 1-7 possess two different hCV identification numbers,as follows:

-   -   hCV1129436 is equivalent to hCV26581155    -   hCV15954277 is equivalent to hCV22272408    -   hCV16173091 is equivalent to hCV25473098    -   hCV16179628 is equivalent to hCV22272980    -   hCV16195242 is equivalent to hCV22274712    -   hCV7482175 is equivalent to hCV26546221

Column heading Definition Marker Internal hCV identification number forthe tested SNP Gene Name HUGO gene symbol for the gene in which the SNPresides Sample Set Sample set used in the analysis (“Sample Set 1” or“Sample Set 2”) p-value The result of the asymptotic chi square test forallelic, dominant, or recessive (based on the genotype reported in the“Mode” column) genotypic association, or the results of Armitagetrendtest for additive genotypic association. For SNPs for whichinformation is provided in an “Adjust” column, it is the result ofallelic, additive, dominant, or recessive (based on the genotypereported in the “Mode” column) p-value of the stratified analysis withCochran Mantel Haenszel test. OR Allelic, dominant, recessive, oradditive (based on the genotype reported in Mode column) odds ratio 95%CI 95% confidence interval of the OR reported Case Freq. Frequency ofAllele1, or genotype containing Allele1, in the case group Cntrl Freq.Frequency of Allele1, or genotype containing Allele1, in the controlgroup Allele1 Nucleotide (allele) of the tested SNP for which statisticsare being reported Mode Mode of inheritance for which p-values arereported: Dom: dominant Rec: recessive Add: Additive Allelic StrataStratum in which the association study analysis was based All:unstratified M: in male F: in female Age T1: age tertile 1 Age T2: agetertile 2 Age T3: age tertile 3 Smoke+: people who are past or currentsmoker Smoke−: people who never smoked MI−: people without heart attackAdjust Adjustments that were done for the Cochran Mantel Haenszel test(Indicates that the p-value was determined using a Cochran MantelHaenszel test that was adjusted for confounders)

DESCRIPTION OF THE FIGURE

FIG. 1 provides a diagrammatic representation of a computer-baseddiscovery system containing the SNP information of the present inventionin computer readable form.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides SNPs associated with stenosis, nucleicacid molecules containing SNPs, methods and reagents for the detectionof the SNPs disclosed herein, uses of these SNPs for the development ofdetection reagents, and assays or kits that utilize such reagents. Thestenosis-associated SNPs disclosed herein are useful for diagnosing,screening for, and evaluating predisposition to stenosis and relatedpathologies in humans. Furthermore, such SNPs and their encoded productsare useful targets for the development of therapeutic agents.

A large number of SNPs have been identified from re-sequencing DNA from39 individuals, and they are indicated as “Applera” SNP source in Tables1-2. Their allele frequencies observed in each of the Caucasian andAfrican-American ethnic groups are provided. Additional SNPs includedherein were previously identified during shotgun sequencing and assemblyof the human genome, and they are indicated as “Celera” SNP source inTables 1-2. Furthermore, the information provided in Table 1-2,particularly the allele frequency information obtained from 39individuals and the identification of the precise position of each SNPwithin each gene/transcript, allows haplotypes (i.e., groups of SNPsthat are co-inherited) to be readily inferred. The present inventionencompasses SNP haplotypes, as well as individual SNPs.

Thus, the present invention provides individual SNPs associated withstenosis, as well as combinations of SNPs and haplotypes in geneticregions associated with stenosis, polymorphic/variant transcriptsequences (SEQ ID NOS:1-284) and genomic sequences (SEQ IDNOS:5608-5780) containing SNPs, encoded amino acid sequences (SEQ IDNOS: 285-568), and both transcript-based SNP context sequences (SEQ IDNOS: 569-5607) and genomic-based SNP context sequences (SEQ IDNOS:5781-26,130) (transcript sequences, protein sequences, andtranscript-based SNP context sequences are provided in Table 1 and theSequence Listing; genomic sequences and genomic-based SNP contextsequences are provided in Table 2 and the Sequence Listing), methods ofdetecting these polymorphisms in a test sample, methods of determiningthe risk of an individual of having or developing stenosis, methods ofscreening for compounds useful for treating disorders associated with avariant gene/protein such as stenosis, compounds identified by thesescreening methods, methods of using the disclosed SNPs to select atreatment strategy, methods of treating a disorder associated with avariant gene/protein (i.e., therapeutic methods), and methods of usingthe SNPs of the present invention for human identification.

The present invention provides novel SNPs associated with stenosis, aswell as SNPs that were previously known in the art, but were notpreviously known to be associated with stenosis. Accordingly, thepresent invention provides novel compositions and methods based on thenovel SNPs disclosed herein, and also provides novel methods of usingthe known, but previously unassociated, SNPs in methods relating tostenosis (e.g., for diagnosing stenosis, etc.). In Tables 1-2, knownSNPs are identified based on the public database in which they have beenobserved, which is indicated as one or more of the following SNP types:“dbSNP”=SNP observed in dbSNP, “HGBASE”=SNP observed in HGBASE, and“HGMD”=SNP observed in the Human Gene Mutation Database (HGMD). NovelSNPs for which the SNP source is only “Applera” and none other, i.e.,those that have not been observed in any public databases and which werealso not observed during shotgun sequencing and assembly of the Celerahuman genome sequence (i.e., “Celera” SNP source), are indicated inTables 3-4.

Particular SNP alleles of the present invention can be associated witheither an increased risk of having or developing stenosis, or adecreased risk of having or developing stenosis. SNP alleles that areassociated with a decreased risk of having or developing stenosis may bereferred to as “protective” alleles, and SNP alleles that are associatedwith an increased risk of having or developing stenosis may be referredto as “susceptibility” alleles or “risk factors”. Thus, whereas certainSNPs (or their encoded products) can be assayed to determine whether anindividual possesses a SNP allele that is indicative of an increasedrisk of having or developing stenosis (i.e., a susceptibility allele),other SNPs (or their encoded products) can be assayed to determinewhether an individual possesses a SNP allele that is indicative of adecreased risk of having or developing stenosis (i.e., a protectiveallele). Similarly, particular SNP alleles of the present invention canbe associated with either an increased or decreased likelihood ofresponding to a particular treatment or therapeutic compound, or anincreased or decreased likelihood of experiencing toxic effects from aparticular treatment or therapeutic compound. The term “altered” may beused herein to encompass either of these two possibilities (e.g., anincreased or a decreased risk/likelihood).

Those skilled in the art will readily recognize that nucleic acidmolecules may be double-stranded molecules and that reference to aparticular site on one strand refers, as well, to the corresponding siteon a complementary strand. In defining a SNP position, SNP allele, ornucleotide sequence, reference to an adenine, a thymine (uridine), acytosine, or a guanine at a particular site on one strand of a nucleicacid molecule also defines the thymine (uridine), adenine, guanine, orcytosine (respectively) at the corresponding site on a complementarystrand of the nucleic acid molecule. Thus, reference may be made toeither strand in order to refer to a particular SNP position, SNPallele, or nucleotide sequence. Probes and primers, may be designed tohybridize to either strand and SNP genotyping methods disclosed hereinmay generally target either strand. Throughout the specification, inidentifying a SNP position, reference is generally made to theprotein-encoding strand, only for the purpose of convenience.

References to variant peptides, polypeptides, or proteins of the presentinvention include peptides, polypeptides, proteins, or fragmentsthereof, that contain at least one amino acid residue that differs fromthe corresponding amino acid sequence of the art-knownpeptide/polypeptide/protein (the art-known protein may beinterchangeably referred to as the “wild-type”, “reference”, or “normal”protein). Such variant peptides/polypeptides/proteins can result from acodon change caused by a nonsynonymous nucleotide substitution at aprotein-coding SNP position (i.e., a missense mutation) disclosed by thepresent invention. Variant peptides/polypeptides/proteins of the presentinvention can also result from a nonsense mutation, i.e. a SNP thatcreates a premature stop codon, a SNP that generates a read-throughmutation by abolishing a stop codon, or due to any SNP disclosed by thepresent invention that otherwise alters the structure,function/activity, or expression of a protein, such as a SNP in aregulatory region (e.g. a promoter or enhancer) or a SNP that leads toalternative or defective splicing, such as a SNP in an intron or a SNPat an exon/intron boundary. As used herein, the terms “polypeptide”,“peptide”, and “protein” are used interchangeably.

Isolated Nucleic Acid Molecules and SNP Detection Reagents & Kits

Tables 1 and 2 provide a variety of information about each SNP of thepresent invention that is associated with stenosis, including thetranscript sequences (SEQ ID NOS:1-284), genomic sequences (SEQ IDNOS:5608-5780), and protein sequences (SEQ ID NOS:285-568) of theencoded gene products (with the SNPs indicated by IUB codes in thenucleic acid sequences). In addition, Tables 1 and 2 include SNP contextsequences, which generally include 100 nucleotide upstream (5′) plus 100nucleotides downstream (3′) of each SNP position (SEQ ID NOS:569-5607correspond to transcript-based SNP context sequences disclosed in Table1, and SEQ ID NOS:5781-26,130 correspond to genomic-based contextsequences disclosed in Table 2), the alternative nucleotides (alleles)at each SNP position, and additional information about the variant whererelevant, such as SNP type (coding, missense, splice site, UTR, etc.),human populations in which the SNP was observed, observed allelefrequencies, information about the encoded protein, etc.

Isolated Nucleic Acid Molecules

The present invention provides isolated nucleic acid molecules thatcontain one or more SNPs disclosed Table 1 and/or Table 2. Preferredisolated nucleic acid molecules contain one or more SNPs identified inTable 3 and/or Table 4. Isolated nucleic acid molecules containing oneor more SNPs disclosed in at least one of Tables 1-4 may beinterchangeably referred to throughout the present text as“SNP-containing nucleic acid molecules”. Isolated nucleic acid moleculesmay optionally encode a full-length variant protein or fragment thereof.The isolated nucleic acid molecules of the present invention alsoinclude probes and primers (which are described in greater detail belowin the section entitled “SNP Detection Reagents”), which may be used forassaying the disclosed SNPs, and isolated full-length genes,transcripts, cDNA molecules, and fragments thereof, which may be usedfor such purposes as expressing an encoded protein.

As used herein, an “isolated nucleic acid molecule” generally is onethat contains a SNP of the present invention or one that hybridizes tosuch molecule such as a nucleic acid with a complementary sequence, andis separated from most other nucleic acids present in the natural sourceof the nucleic acid molecule. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule containing a SNP of the presentinvention, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. A nucleicacid molecule can be fused to other coding or regulatory sequences andstill be considered “isolated”. Nucleic acid molecules present innon-human transgenic animals, which do not naturally occur in theanimal, are also considered “isolated”. For example, recombinant DNAmolecules contained in a vector are considered “isolated”. Furtherexamples of “isolated” DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells, and purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the isolated SNP-containing DNAmolecules of the present invention. Isolated nucleic acid moleculesaccording to the present invention further include such moleculesproduced synthetically.

Generally, an isolated SNP-containing nucleic acid molecule comprisesone or more SNP positions disclosed by the present invention withflanking nucleotide sequences on either side of the SNP positions. Aflanking sequence can include nucleotide residues that are naturallyassociated with the SNP site and/or heterologous nucleotide sequences.Preferably the flanking sequence is up to about 500, 300, 100, 60, 50,30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in-between)on either side of a SNP position, or as long as the full-length gene orentire protein-coding sequence (or any portion thereof such as an exon),especially if the SNP-containing nucleic acid molecule is to be used toproduce a protein or protein fragment.

For full-length genes and entire protein-coding sequences, a SNPflanking sequence can be, for example, up to about 5 KB, 4 KB, 3 KB, 2KB, 1 KB on either side of the SNP. Furthermore, in such instances, theisolated nucleic acid molecule comprises exonic sequences (includingprotein-coding and/or non-coding exonic sequences), but may also includeintronic sequences. Thus, any protein coding sequence may be eithercontiguous or separated by introns. The important point is that thenucleic acid is isolated from remote and unimportant flanking sequencesand is of appropriate length such that it can be subjected to thespecific manipulations or uses described herein such as recombinantprotein expression, preparation of probes and primers for assaying theSNP position, and other uses specific to the SNP-containing nucleic acidsequences.

An isolated SNP-containing nucleic acid molecule can comprise, forexample, a full-length gene or transcript, such as a gene isolated fromgenomic DNA (e.g., by cloning or PCR amplification), a cDNA molecule, oran mRNA transcript molecule. Polymorphic transcript sequences areprovided in Table 1 and in the Sequence Listing (SEQ ID NOS:1-284), andpolymorphic genomic sequences are provided in Table 2 and in theSequence Listing (SEQ ID NOS:5608-5780). Furthermore, fragments of suchfull-length genes and transcripts that contain one or more SNPsdisclosed herein are also encompassed by the present invention, and suchfragments may be used, for example, to express any part of a protein,such as a particular functional domain or an antigenic epitope.

Thus, the present invention also encompasses fragments of the nucleicacid sequences provided in Tables 1-2 (transcript sequences are providedin Table 1 as SEQ ID NOS:1-284, genomic sequences are provided in Table2 as SEQ ID NOS:5608-5780, transcript-based SNP context sequences areprovided in Table 1 as SEQ ID NO:569-5607, and genomic-based SNP contextsequences are provided in Table 2 as SEQ ID NO:5781-26,130) and theircomplements. A fragment typically comprises a contiguous nucleotidesequence at least about 8 or more nucleotides, more preferably at leastabout 12 or more nucleotides, and even more preferably at least about 16or more nucleotides. Further, a fragment could comprise at least about18, 20, 22, 25, 30, 40, 50, 60, 100, 250 or 500 (or any other numberin-between) nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encodeepitope-bearing regions of a variant peptide or regions of a variantpeptide that differ from the normal/wild-type protein, or can be usefulas a polynucleotide probe or primer. Such fragments can be isolatedusing the nucleotide sequences provided in Table 1 and/or Table 2 forthe synthesis of a polynucleotide probe. A labeled probe can then beused, for example, to screen a cDNA library, genomic DNA library, ormRNA to isolate nucleic acid corresponding to the coding region.Further, primers can be used in amplification reactions, such as forpurposes of assaying one or more SNPs sites or for cloning specificregions of a gene.

An isolated nucleic acid molecule of the present invention furtherencompasses a SNP-containing polynucleotide that is the product of anyone of a variety of nucleic acid amplification methods, which are usedto increase the copy numbers of a polynucleotide of interest in anucleic acid sample. Such amplification methods are well known in theart, and they include but are not limited to, polymerase chain reaction(PCR) (U.S. Pat. Nos. 4,683,195; and 4,683,202; PCR Technology:Principles and Applications for DNA Amplification, ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992), ligase chain reaction (LCR) (Wu andWallace, Genomics 4:560, 1989; Landegren et al., Science 241:1077,1988), strand displacement amplification (SDA) (U.S. Pat. Nos.5,270,184; and 5,422,252), transcription-mediated amplification (TMA)(U.S. Pat. No. 5,399,491), linked linear amplification (LLA) (U.S. Pat.No. 6,027,923), and the like, and isothermal amplification methods suchas nucleic acid sequence based amplification (NASBA), and self-sustainedsequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990). Based on such methodologies, a person skilled in the artcan readily design primers in any suitable regions 5′ and 3′ to a SNPdisclosed herein. Such primers may be used to amplify DNA of any lengthso long that it contains the SNP of interest in its sequence.

As used herein, an “amplified polynucleotide” of the invention is aSNP-containing nucleic acid molecule whose amount has been increased atleast two fold by any nucleic acid amplification method performed invitro as compared to its starting amount in a test sample. In otherpreferred embodiments, an amplified polynucleotide is the result of atleast ten fold, fifty fold, one hundred fold, one thousand fold, or eventen thousand fold increase as compared to its starting amount in a testsample. In a typical PCR amplification, a polynucleotide of interest isoften amplified at least fifty thousand fold in amount over theunamplified genomic DNA, but the precise amount of amplification neededfor an assay depends on the sensitivity of the subsequent detectionmethod used.

Generally, an amplified polynucleotide is at least about 16 nucleotidesin length. More typically, an amplified polynucleotide is at least about20 nucleotides in length. In a preferred embodiment of the invention, anamplified polynucleotide is at least about 30 nucleotides in length. Ina more preferred embodiment of the invention, an amplifiedpolynucleotide is at least about 32, 40, 45, 50, or 60 nucleotides inlength. In yet another preferred embodiment of the invention, anamplified polynucleotide is at least about 100, 200, or 300 nucleotidesin length. While the total length of an amplified polynucleotide of theinvention can be as long as an exon, an intron or the entire gene wherethe SNP of interest resides, an amplified product is typically nogreater than about 1,000 nucleotides in length (although certainamplification methods may generate amplified products greater than 1000nucleotides in length). More preferably, an amplified polynucleotide isnot greater than about 600 nucleotides in length. It is understood thatirrespective of the length of an amplified polynucleotide, a SNP ofinterest may be located anywhere along its sequence.

In a specific embodiment of the invention, the amplified product is atleast about 0.201 nucleotides in length, comprises one of thetranscript-based context sequences or the genomic-based contextsequences shown in Tables 1-2. Such a product may have additionalsequences on its 5′ end or 3′ end or both. In another embodiment, theamplified product is about 101 nucleotides in length, and it contains aSNP disclosed herein. Preferably, the SNP is located at the middle ofthe amplified product (e.g., at position 101 in an amplified productthat is 201 nucleotides in length, or at position 51 in an amplifiedproduct that is 101 nucleotides in length), or within 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 15, or 20 nucleotides from the middle of the amplifiedproduct (however, as indicated above, the SNP of interest may be locatedanywhere along the length of the amplified product).

The present invention provides isolated nucleic acid molecules thatcomprise, consist of, or consist essentially of one or morepolynucleotide sequences that contain one or more SNPs disclosed herein,complements thereof, and SNP-containing fragments thereof.

Accordingly, the present invention provides nucleic acid molecules thatconsist of any of the nucleotide sequences shown in Table 1 and/or Table2 (transcript sequences are provided in Table 1 as SEQ ID NOS:1-284,genomic sequences are provided in Table 2 as SEQ ID NOS:5608-5780,transcript-based SNP context sequences are provided in Table 1 as SEQ IDNO:569-5607, and genomic-based SNP context sequences are provided inTable 2 as SEQ ID NO:5781-26,130), or any nucleic acid molecule thatencodes any of the variant proteins provided in Table 1 (SEQ IDNOS:285-568). A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of any of the nucleotide sequences shown in Table 1and/or Table 2 (transcript sequences are provided in Table 1 as SEQ IDNOS:1-284, genomic sequences are provided in Table 2 as SEQ IDNOS:5608-5780, transcript-based SNP context sequences are provided inTable 1 as SEQ ID NO:569-5607, and genomic-based SNP context sequencesare provided in Table 2 as SEQ ID NO:5781-26,130), or any nucleic acidmolecule that encodes any of the variant proteins provided in Table 1(SEQ ID NOS:285-568). A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleotide residues in the final nucleic acid molecule.

The present invention further provides nucleic acid molecules thatcomprise any of the nucleotide sequences shown in Table 1 and/or Table 2or a SNP-containing fragment thereof (transcript sequences are providedin Table 1 as SEQ ID NOS:1-284, genomic sequences are provided in Table2 as SEQ ID NOS:5608-5780, transcript-based SNP context sequences areprovided in Table 1 as SEQ ID NO:569-5607, and genomic-based SNP contextsequences are provided in Table 2 as SEQ ID NO:5781-26,130), or anynucleic acid molecule that encodes any of the variant proteins providedin Table 1 (SEQ ID NOS:285-568). A nucleic acid molecule comprises anucleotide sequence when the nucleotide sequence is at least part of thefinal nucleotide sequence of the nucleic acid molecule. In such afashion, the nucleic acid molecule can be only the nucleotide sequenceor have additional nucleotide residues, such as residues that arenaturally associated with it or heterologous nucleotide sequences. Sucha nucleic acid molecule can have one to a few additional nucleotides orcan comprise many more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readily madeand isolated is provided below, and such techniques are well known tothose of ordinary skill in the art (Sambrook and Russell, 2000,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY).

The isolated nucleic acid molecules can encode mature proteins plusadditional amino or carboxyl-terminal amino acids or both, or aminoacids interior to the mature peptide (when the mature form has more thanone peptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life, or facilitatemanipulation of a protein for assay or production. As generally is thecase in situ, the additional amino acids may be processed away from themature protein by cellular enzymes.

Thus, the isolated nucleic acid molecules include, but are not limitedto, nucleic acid molecules having a sequence encoding a peptide alone, asequence encoding a mature peptide and additional coding sequences suchas a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), a sequence encoding a mature peptide with or withoutadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut untranslated sequences that play a role in, for example,transcription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding, and/or stability of mRNA. In addition, thenucleic acid molecules may be fused to heterologous marker sequencesencoding, for example, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA, which may beobtained, for example, by molecular cloning or produced by chemicalsynthetic techniques or by a combination thereof (Sambrook and Russell,2000, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,NY). Furthermore, isolated nucleic acid molecules, particularly SNPdetection reagents such as probes and primers, can also be partially orcompletely in the form of one or more types of nucleic acid analogs,such as peptide nucleic acid (PNA) (U.S. Pat. Nos. 5,539,082; 5,527,675;5,623,049; 5,714,331). The nucleic acid, especially DNA, can bedouble-stranded or single-stranded. Single-stranded nucleic acid can bethe coding strand (sense strand) or the complementary non-coding strand(anti-sense strand). DNA, RNA, or PNA segments can be assembled, forexample, from fragments of the human genome (in the case of DNA or RNA)or single nucleotides, short oligonucleotide linkers, or from a seriesof oligonucleotides, to provide a synthetic nucleic acid molecule.Nucleic acid molecules can be readily synthesized using the sequencesprovided herein as a reference; oligonucleotide and PNA oligomersynthesis techniques are well known in the art (see, e.g., Corey,“Peptide nucleic acids: expanding the scope of nucleic acidrecognition”, Trends Biotechnol. 1997 June; 15(6):224-9, and Hyrup etal., “Peptide nucleic acids (PNA): synthesis, properties and potentialapplications”, Bioorg Med Chem. 1996 January; 4(1):5-23). Furthermore,large-scale automated oligonucleotide/PNA synthesis (including synthesison an array or bead surface or other solid support) can readily beaccomplished using commercially available nucleic acid synthesizers,such as the Applied Biosystems (Foster City, Calif.) 3900High-Throughput DNA Synthesizer or Expedite 8909 Nucleic Acid SynthesisSystem, and the sequence information provided herein.

The present invention encompasses nucleic acid analogs that containmodified, synthetic, or non-naturally occurring nucleotides orstructural elements or other alternative/modified nucleic acidchemistries known in the art. Such nucleic acid analogs are useful, forexample, as detection reagents (e.g., primers/probes) for detecting oneor more SNPs identified in Table 1 and/or Table 2. Furthermore,kits/systems (such as beads, arrays, etc.) that include these analogsare also encompassed by the present invention. For example, PNAoligomers that are based on the polymorphic sequences of the presentinvention are specifically contemplated. PNA oligomers are analogs ofDNA in which the phosphate backbone is replaced with a peptide-likebackbone (Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters,4: 1081-1082 (1994), Petersen et al., Bioorganic & Medicinal ChemistryLetters, 6: 793-796 (1996), Kumar et al., Organic Letters 3(9):1269-1272 (2001), WO96/04000). PNA hybridizes to complementary RNA orDNA with higher affinity and specificity than conventionaloligonucleotides and oligonucleotide analogs. The properties of PNAenable novel molecular biology and biochemistry applicationsunachievable with traditional oligonucleotides and peptides.

Additional examples of nucleic acid modifications that improve thebinding properties and/or stability of a nucleic acid include the use ofbase analogs such as inosine, intercalators (U.S. Pat. No. 4,835,263)and the minor groove binders (U.S. Pat. No. 5,801,115). Thus, referencesherein to nucleic acid molecules, SNP-containing nucleic acid molecules,SNP detection reagents (e.g., probes and primers),oligonucleotides/polynucleotides include PNA oligomers and other nucleicacid analogs. Other examples of nucleic acid analogs andalternative/modified nucleic acid chemistries known in the art aredescribed in Current Protocols in Nucleic Acid Chemistry, John Wiley &Sons, N.Y. (2002).

The present invention further provides nucleic acid molecules thatencode fragments of the variant polypeptides disclosed herein as well asnucleic acid molecules that encode obvious variants of such variantpolypeptides. Such nucleic acid molecules may be naturally occurring,such as paralogs (different locus) and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Non-naturally occurring variants may be made by mutagenesistechniques, including those applied to nucleic acid molecules, cells, ororganisms. Accordingly, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions (in addition to theSNPs disclosed in Tables 1-2). Variation can occur in either or both thecoding and non-coding regions. The variations can produce conservativeand/or non-conservative amino acid substitutions.

Further variants of the nucleic acid molecules disclosed in Tables 1-2,such as naturally occurring allelic variants (as well as orthologs andparalogs) and synthetic variants produced by mutagenesis techniques, canbe identified and/or produced using methods well known in the art. Suchfurther variants can comprise a nucleotide sequence that shares at least70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity with a nucleic acid sequence disclosed in Table 1and/or Table 2 (or a fragment thereof) and that includes a novel SNPallele disclosed in Table 1 and/or Table 2. Further, variants cancomprise a nucleotide sequence that encodes a polypeptide that shares atleast 70-80%, 80-85%, 85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity with a polypeptide sequence disclosed in Table 1(or a fragment thereof) and that includes a novel SNP allele disclosedin Table 1 and/or Table 2. Thus, the present invention specificallycontemplates isolated nucleic acid molecule that have a certain degreeof sequence variation compared with the sequences shown in Tables 1-2,but that contain a novel SNP allele disclosed herein. In other words, aslong as an isolated nucleic acid molecule contains a novel SNP alleledisclosed herein, other portions of the nucleic acid molecule that flankthe novel SNP allele can vary to some degree from the specifictranscript, genomic, and context sequences shown in Tables 1-2, and canencode a polypeptide that varies to some degree from the specificpolypeptide sequences shown in Table 1.

To determine the percent identity of two amino acid sequences or twonucleotide sequences of two molecules that share sequence homology, thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). In a preferred embodiment, atleast 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of areference sequence is aligned for comparison purposes. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein, amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). In a preferred embodiment, the percent identity between two aminoacid sequences is determined using the Needleman and Wunsch algorithm(J. Mol. Biol. (48):444-453 (1970)) which has been incorporated into theGAP program in the GCG software package, using either a Blossom 62matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or4 and a length weight of 1, 2, 3, 4, 5, or 6.

In yet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1):387(1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4.

The nucleotide and amino acid sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. In addition to BLAST, examples of othersearch and sequence comparison programs used in the art include, but arenot limited to, FASTA (Pearson, Methods Mol. Biol. 25, 365-389 (1994))and KERR (Dufresne et al., Nat Biotechnol 2002 December; 20(12):1269-71). For further information regarding bioinformatics techniques,see Current Protocols in Bioinformatics, John Wiley & Sons, Inc., N.Y.

The present invention further provides non-coding fragments of thenucleic acid molecules disclosed in Table 1 and/or Table 2. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, intronic sequences, 5′ untranslatedregions (UTRs), 3′ untranslated regions, gene modulating sequences andgene termination sequences. Such fragments are useful, for example, incontrolling heterologous gene expression and in developing screens toidentify gene-modulating agents.

SNP Detection Reagents

In a specific aspect of the present invention, the SNPs disclosed inTable 1 and/or Table 2, and their associated transcript sequences(provided in Table 1 as SEQ ID NOS:1-284), genomic sequences (providedin Table 2 as SEQ ID NOS:5608-5780), and context sequences(transcript-based context sequences are provided in Table 1 as SEQ IDNOS:569-5607; genomic-based context sequences are provided in Table 2 asSEQ ID NOS:5781-26,130), can be used for the design of SNP detectionreagents. As used herein, a “SNP detection reagent” is a reagent thatspecifically detects a specific target SNP position disclosed herein,and that is preferably specific for a particular nucleotide (allele) ofthe target SNP position (i.e., the detection reagent preferably candifferentiate between different alternative nucleotides at a target SNPposition, thereby allowing the identity of the nucleotide present at thetarget SNP position to be determined). Typically, such detection reagenthybridizes to a target SNP-containing nucleic acid molecule bycomplementary base-pairing in a sequence specific manner, anddiscriminates the target variant sequence from other nucleic acidsequences such as an art-known form in a test sample. An example of adetection reagent is a probe that hybridizes to a target nucleic acidcontaining one or more of the SNPs provided in Table 1 and/or Table 2.In a preferred embodiment, such a probe can differentiate betweennucleic acids having a particular nucleotide (allele) at a target SNPposition from other nucleic acids that have a different nucleotide atthe same target SNP position. In addition, a detection reagent mayhybridize to a specific region 5′ and/or 3′ to a SNP position,particularly a region corresponding to the context sequences provided inTable 1 and/or Table 2 (transcript-based context sequences are providedin Table 1 as SEQ ID NOS:569-5607; genomic-based context sequences areprovided in Table 2 as SEQ ID NOS:5781-26,130). Another example of adetection reagent is a primer which acts as an initiation point ofnucleotide extension along a complementary strand of a targetpolynucleotide. The SNP sequence information provided herein is alsouseful for designing primers, e.g. allele-specific primers, to amplify(e.g., using PCR) any SNP of the present invention.

In one preferred embodiment of the invention, a SNP detection reagent isan isolated or synthetic DNA or RNA polynucleotide probe or primer orPNA oligomer, or a combination of DNA, RNA and/or PNA, that hybridizesto a segment of a target nucleic acid molecule containing a SNPidentified in Table 1 and/or Table 2. A detection reagent in the form ofa polynucleotide may optionally contain modified base analogs,intercalators or minor groove binders. Multiple detection reagents suchas probes may be, for example, affixed to a solid support (e.g., arraysor beads) or supplied in solution (e.g., probe/primer sets for enzymaticreactions such as PCR, RT-PCR, TaqMan assays, or primer-extensionreactions) to form a SNP detection kit.

A probe or primer typically is a substantially purified oligonucleotideor PNA oligomer. Such oligonucleotide typically comprises a region ofcomplementary nucleotide sequence that hybridizes under stringentconditions to at least about 8, 10, 12, 16, 18, 20, 22, 25, 30, 40, 50,60, 100 (or any other number in-between) or more consecutive nucleotidesin a target nucleic acid molecule. Depending on the particular assay,the consecutive nucleotides can either include the target SNP position,or be a specific region in close enough proximity 5′ and/or 3′ to theSNP position to carry out the desired assay.

Other preferred primer and probe sequences can readily be determinedusing the transcript sequences (SEQ ID NOS:1-284), genomic sequences(SEQ ID NOS:5608-5780), and SNP context sequences (transcript-basedcontext sequences are provided in Table 1 as SEQ ID NOS:569-5607;genomic-based context sequences are provided in Table 2 as SEQ IDNOS:5781-26,130) disclosed in the Sequence Listing and in Tables 1-2. Itwill be apparent to one of skill in the art that such primers and probesare directly useful as reagents for genotyping the SNPs of the presentinvention, and can be incorporated into any kit/system format.

In order to produce a probe or primer specific for a targetSNP-containing sequence, the gene/transcript and/or context sequencesurrounding the SNP of interest is typically examined using a computeralgorithm which starts at the 5′ or at the 3′ end of the nucleotidesequence. Typical algorithms will then identify oligomers of definedlength that are unique to the gene/SNP context sequence, have a GCcontent within a range suitable for hybridization, lack predictedsecondary structure that may interfere with hybridization, and/orpossess other desired characteristics or that lack other undesiredcharacteristics.

A primer or probe of the present invention is typically at least about 8nucleotides in length. In one embodiment of the invention, a primer or aprobe is at least about 10 nucleotides in length. In a preferredembodiment, a primer or a probe is at least about 12 nucleotides inlength. In a more preferred embodiment, a primer or probe is at leastabout 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.While the maximal length of a probe can be as long as the targetsequence to be detected, depending on the type of assay in which it isemployed, it is typically less than about 50, 60, 65, or 70 nucleotidesin length. In the case of a primer, it is typically less than about 30nucleotides in length. In a specific preferred embodiment of theinvention, a primer or a probe is within the length of about 18 andabout 28 nucleotides. However, in other embodiments, such as nucleicacid arrays and other embodiments in which probes are affixed to asubstrate, the probes can be longer, such as on the order of 30-70, 75,80, 90, 100, or more nucleotides in length (see the section belowentitled “SNP Detection Kits and Systems”).

For analyzing SNPs, it may be appropriate to use oligonucleotidesspecific for alternative SNP alleles. Such oligonucleotides which detectsingle nucleotide variations in target sequences may be referred to bysuch terms as “allele-specific oligonucleotides”, “allele-specificprobes”, or “allele-specific primers”. The design and use ofallele-specific probes for analyzing polymorphisms is described in,e.g., Mutation Detection A Practical Approach, ed. Cotton et al. OxfordUniversity Press, 1998; Saiki et al., Nature 324, 163-166 (1986);Dattagupta, EP235,726; and Saiki, WO 89/11548.

While the design of each allele-specific primer or probe depends onvariables such as the precise composition of the nucleotide sequencesflanking a SNP position in a target nucleic acid molecule, and thelength of the primer or probe, another factor in the use of primers andprobes is the stringency of the condition under which the hybridizationbetween the probe or primer and the target sequence is performed. Higherstringency conditions utilize buffers with lower ionic strength and/or ahigher reaction temperature, and tend to require a more perfect matchbetween probe/primer and a target sequence in order to form a stableduplex. If the stringency is too high, however, hybridization may notoccur at all. In contrast, lower stringency conditions utilize bufferswith higher ionic strength and/or a lower reaction temperature, andpermit the formation of stable duplexes with more mismatched basesbetween a probe/primer and a target sequence. By way of example and notlimitation, exemplary conditions for high stringency hybridizationconditions using an allele-specific probe are as follows:Prehybridization with a solution containing 5× standard saline phosphateEDTA (SSPE), 0.5% NaDodSO₄ (SDS) at 55° C., and incubating probe withtarget nucleic acid molecules in the same solution at the sametemperature, followed by washing with a solution containing 2×SSPE, and0.1% SDS at 55° C. or room temperature.

Moderate stringency hybridization conditions may be used forallele-specific primer extension reactions with a solution containing,e.g., about 50 mM KCl at about 46° C. Alternatively, the reaction may becarried out at an elevated temperature such as 60° C. In anotherembodiment, a moderately stringent hybridization condition suitable foroligonucleotide ligation assay (OLA) reactions wherein two probes areligated if they are completely complementary to the target sequence mayutilize a solution of about 100 mM KCl at a temperature of 46° C.

In a hybridization-based assay, allele-specific probes can be designedthat hybridize to a segment of target DNA from one individual but do nothybridize to the corresponding segment from another individual due tothe presence of different polymorphic forms (e.g., alternative SNPalleles/nucleotides) in the respective DNA segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant detectable difference in hybridizationintensity between alleles, and preferably an essentially binaryresponse, whereby a probe hybridizes to only one of the alleles orsignificantly more strongly to one allele. While a probe may be designedto hybridize to a target sequence that contains a SNP site such that theSNP site aligns anywhere along the sequence of the probe, the probe ispreferably designed to hybridize to a segment of the target sequencesuch that the SNP site aligns with a central position of the probe(e.g., a position within the probe that is at least three nucleotidesfrom either end of the probe). This design of probe generally achievesgood discrimination in hybridization between different allelic forms.

In another embodiment, a probe or primer may be designed to hybridize toa segment of target DNA such that the SNP aligns with either the 5′ mostend or the 3′ most end of the probe or primer. In a specific preferredembodiment which is particularly suitable for use in a oligonucleotideligation assay (U.S. Pat. No. 4,988,617), the 3′ most nucleotide of theprobe aligns with the SNP position in the target sequence.

Oligonucleotide probes and primers may be prepared by methods well knownin the art. Chemical synthetic methods include, but are limited to, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology 68:90; the phosphodiester method described by Brown et al.,1979, Methods in Enzymology 68:109, the diethylphosphoamidate methoddescribed by Beaucage et al., 1981, Tetrahedron Letters 22:1859; and thesolid support method described in U.S. Pat. No. 4,458,066.

Allele-specific probes are often used in pairs (or, less commonly, insets of 3 or 4, such as if a SNP position is known to have 3 or 4alleles, respectively, or to assay both strands of a nucleic acidmolecule for a target SNP allele), and such pairs may be identicalexcept for a one nucleotide mismatch that represents the allelicvariants at the SNP position.

Commonly, one member of a pair perfectly matches a reference form of atarget sequence that has a more common SNP allele (i.e., the allele thatis more frequent in the target population) and the other member of thepair perfectly matches a form of the target sequence that has a lesscommon SNP allele (i.e., the allele that is rarer in the targetpopulation). In the case of an array, multiple pairs of probes can beimmobilized on the same support for simultaneous analysis of multipledifferent polymorphisms.

In one type of PCR-based assay, an allele-specific primer hybridizes toa region on a target nucleic acid molecule that overlaps a SNP positionand only primes amplification of an allelic form to which the primerexhibits perfect complementarity (Gibbs, 1989, Nucleic Acid Res. 172427-2448). Typically, the primer's 3′-most nucleotide is aligned withand complementary to the SNP position of the target nucleic acidmolecule. This primer is used in conjunction with a second primer thathybridizes at a distal site. Amplification proceeds from the twoprimers, producing a detectable product that indicates which allelicform is present in the test sample. A control is usually performed witha second pair of primers, one of which shows a single base mismatch atthe polymorphic site and the other of which exhibits perfectcomplementarity to a distal site. The single-base mismatch preventsamplification or substantially reduces amplification efficiency, so thateither no detectable product is formed or it is formed in lower amountsor at a slower pace. The method generally works most effectively whenthe mismatch is at the 3′-most position of the oligonucleotide (i.e.,the 3′-most position of the oligonucleotide aligns with the target SNPposition) because this position is most destabilizing to elongation fromthe primer (see, e.g., WO 93/22456). This PCR-based assay can beutilized as part of the TaqMan assay, described below.

In a specific embodiment of the invention, a primer of the inventioncontains a sequence substantially complementary to a segment of a targetSNP-containing nucleic acid molecule except that the primer has amismatched nucleotide in one of the three nucleotide positions at the3′-most end of the primer, such that the mismatched nucleotide does notbase pair with a particular allele at the SNP site. In a preferredembodiment, the mismatched nucleotide in the primer is the second fromthe last nucleotide at the 3′-most position of the primer. In a morepreferred embodiment, the mismatched nucleotide in the primer is thelast nucleotide at the 3′-most position of the primer.

In another embodiment of the invention, a SNP detection reagent of theinvention is labeled with a fluorogenic reporter dye that emits adetectable signal. While the preferred reporter dye is a fluorescentdye, any reporter dye that can be attached to a detection reagent suchas an oligonucleotide probe or primer is suitable for use in theinvention. Such dyes include, but are not limited to, Acridine, AMCA,BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin,Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine,Rhodol Green, Tamra, Rox, and Texas Red.

In yet another embodiment of the invention, the detection reagent may befurther labeled with a quencher dye such as Tamra, especially when thereagent is used as a self-quenching probe such as a TaqMan (U.S. Pat.Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos.5,118,801 and 5,312,728), or other stemless or linear beacon probe(Livak et al., 1995, PCR Method Appl. 4:357-362; Tyagi et al., 1996,Nature Biotechnology 14: 303-308; Nazarenko et al., 1997, Nucl. AcidsRes. 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635).

The detection reagents of the invention may also contain other labels,including but not limited to, biotin for streptavidin binding, haptenfor antibody binding, and oligonucleotide for binding to anothercomplementary oligonucleotide such as pairs of zipcodes.

The present invention also contemplates reagents that do not contain (orthat are complementary to) a SNP nucleotide identified herein but thatare used to assay one or more SNPs disclosed herein. For example,primers that flank, but do not hybridize directly to a target SNPposition provided herein are useful in primer extension reactions inwhich the primers hybridize to a region adjacent to the target SNPposition (i.e., within one or more nucleotides from the target SNPsite). During the primer extension reaction, a primer is typically notable to extend past a target SNP site if a particular nucleotide(allele) is present at that target SNP site, and the primer extensionproduct can readily be detected in order to determine which SNP alleleis present at the target SNP site. For example, particular ddNTPs aretypically used in the primer extension reaction to terminate primerextension once a ddNTP is incorporated into the extension product (aprimer extension product which includes a ddNTP at the 3′-most end ofthe primer extension product, and in which the ddNTP corresponds to aSNP disclosed herein, is a composition that is encompassed by thepresent invention). Thus, reagents that bind to a nucleic acid moleculein a region adjacent to a SNP site, even though the bound sequences donot necessarily include the SNP site itself, are also encompassed by thepresent invention.

SNP Detection Kits and Systems

A person skilled in the art will recognize that, based on the SNP andassociated sequence information disclosed herein, detection reagents canbe developed and used to assay any SNP of the present inventionindividually or in combination, and such detection reagents can bereadily incorporated into one of the established kit or system formatswhich are well known in the art. The terms “kits” and “systems”, as usedherein in the context of SNP detection reagents, are intended to referto such things as combinations of multiple SNP detection reagents, orone or more SNP detection reagents in combination with one or more othertypes of elements or components (e.g., other types of biochemicalreagents, containers, packages such as packaging intended for commercialsale, substrates to which SNP detection reagents are attached,electronic hardware components, etc.). Accordingly, the presentinvention further provides SNP detection kits and systems, including butnot limited to, packaged probe and primer sets (e.g., TaqManprobe/primer sets), arrays/microarrays of nucleic acid molecules, andbeads that contain one or more probes, primers, or other detectionreagents for detecting one or more SNPs of the present invention. Thekits/systems can optionally include various electronic hardwarecomponents; for example, arrays (“DNA chips”) and microfluidic systems(“lab-on-a-chip” systems) provided by various manufacturers typicallycomprise hardware components. Other kits/systems (e.g., probe/primersets) may not include electronic hardware components, but may becomprised of, for example, one or more SNP detection reagents (alongwith, optionally, other biochemical reagents) packaged in one or morecontainers.

In some embodiments, a SNP detection kit typically contains one or moredetection reagents and other components (e.g., a buffer, enzymes such asDNA polymerases or ligases, chain extension nucleotides such asdeoxynucleotide triphosphates, and in the case of Sanger-type DNAsequencing reactions, chain terminating nucleotides, positive controlsequences, negative control sequences, and the like) necessary to carryout an assay or reaction, such as amplification and/or detection of aSNP-containing nucleic acid molecule. A kit may further contain meansfor determining the amount of a target nucleic acid, and means forcomparing the amount with a standard, and can comprise instructions forusing the kit to detect the SNP-containing nucleic acid molecule ofinterest. In one embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out one or more assays todetect one or more SNPs disclosed herein. In a preferred embodiment ofthe present invention, SNP detection kits/systems are in the form ofnucleic acid arrays, or compartmentalized kits, includingmicrofluidic/lab-on-a-chip systems.

SNP detection kits/systems may contain, for example, one or more probes,or pairs of probes, that hybridize to a nucleic acid molecule at or neareach target SNP position. Multiple pairs of allele-specific probes maybe included in the kit/system to simultaneously assay large numbers ofSNPs, at least one of which is a SNP of the present invention. In somekits/systems, the allele-specific probes are immobilized to a substratesuch as an array or bead. For example, the same substrate can compriseallele-specific probes for detecting at least 1; 10; 100; 1000; 10,000;100,000 (or any other number in-between) or substantially all of theSNPs shown in Table 1 and/or Table 2.

The terms “arrays”, “microarrays”, and “DNA chips” are used hereininterchangeably to refer to an array of distinct polynucleotides affixedto a substrate, such as glass, plastic, paper, nylon or other type ofmembrane, filter, chip, or any other suitable solid support. Thepolynucleotides can be synthesized directly on the substrate, orsynthesized separate from the substrate and then affixed to thesubstrate. In one embodiment, the microarray is prepared and usedaccording to the methods described in U.S. Pat. No. 5,837,832, Chee etal., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al.(1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc.Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated hereinin their entirety by reference. In other embodiments, such arrays areproduced by the methods described by Brown et al., U.S. Pat. No.5,807,522.

Nucleic acid arrays are reviewed in the following references: Zammatteoet al., “New chips for molecular biology and diagnostics”, BiotechnolAnnu Rev. 2002;8:85-101; Sosnowski et al., “Active microelectronic arraysystem for DNA hybridization, genotyping and pharmacogenomicapplications”, Psychiatr Genet. 2002 December; 12(4):181-92; Heller,“DNA microarray technology: devices, systems, and applications”, AnnuRev Biomed Eng. 2002;4:129-53. Epub 2002 Mar. 22; Kolchinsky et al.,“Analysis of SNPs and other genomic variations using gel-based chips”,Hum Mutat. 2002 April; 19(4):343-60; and McGall et al., “High-densitygenechip oligonucleotide probe arrays”, Adv Biochem Eng Biotechnol.2002;77:21-42.

Any number of probes, such as allele-specific probes, may be implementedin an array, and each probe or pair of probes can hybridize to adifferent SNP position. In the case of polynucleotide probes, they canbe synthesized at designated areas (or synthesized separately and thenaffixed to designated areas) on a substrate using a light-directedchemical process. Each DNA chip can contain, for example, thousands tomillions of individual synthetic polynucleotide probes arranged in agrid-like pattern and miniaturized (e.g., to the size of a dime).Preferably, probes are attached to a solid support in an ordered,addressable array.

A microarray can be composed of a large number of unique,single-stranded polynucleotides, usually either synthetic antisensepolynucleotides or fragments of cDNAs, fixed to a solid support. Typicalpolynucleotides are preferably about 6-60 nucleotides in length, morepreferably about 15-30 nucleotides in length, and most preferably about18-25 nucleotides in length. For certain types of microarrays or otherdetection kits/systems, it may be preferable to use oligonucleotidesthat are only about 7-20 nucleotides in length. In other types ofarrays, such as arrays used in conjunction with chemiluminescentdetection technology, preferred probe lengths can be, for example, about15-80 nucleotides in length, preferably about 50-70 nucleotides inlength, more preferably about 55-65 nucleotides in length, and mostpreferably about 60 nucleotides in length. The microarray or detectionkit can contain polynucleotides that cover the known 5′ or 3′ sequenceof a gene/transcript or target SNP site, sequential polynucleotides thatcover the full-length sequence of a gene/transcript; or uniquepolynucleotides selected from particular areas along the length of atarget gene/transcript sequence, particularly areas corresponding to oneor more SNPs disclosed in Table 1 and/or Table 2. Polynucleotides usedin the microarray or detection kit can be specific to a SNP or SNPs ofinterest (e.g., specific to a particular SNP allele at a target SNPsite, or specific to particular SNP alleles at multiple different SNPsites), or specific to a polymorphic gene/transcript orgenes/transcripts of interest.

Hybridization assays based on polynucleotide arrays rely on thedifferences in hybridization stability of the probes to perfectlymatched and mismatched target sequence variants. For SNP genotyping, itis generally preferable that stringency conditions used in hybridizationassays are high enough such that nucleic acid molecules that differ fromone another at as little as a single SNP position can be differentiated(e.g., typical SNP hybridization assays are designed so thathybridization will occur only if one particular nucleotide is present ata SNP position, but will not occur if an alternative nucleotide ispresent at that SNP position). Such high stringency conditions may bepreferable when using, for example, nucleic acid arrays ofallele-specific probes for SNP detection. Such high stringencyconditions are described in the preceding section, and are well known tothose skilled in the art and can be found in, for example, CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6.

In other embodiments, the arrays are used in conjunction withchemiluminescent detection technology. The following patents and patentapplications, which are all hereby incorporated by reference, provideadditional information pertaining to chemiluminescent detection: U.S.patent application Ser. Nos. 10/620,332 and 10/620,333 describechemiluminescent approaches for microarray detection; U.S. Pat. Nos.6,124,478, 6,107,024, 5,994,073, 5,981,768, 5,871,938, 5,843,681,5,800,999, and 5,773,628 describe methods and compositions of dioxetanefor performing chemiluminescent detection; and U.S. publishedapplication US2002/0110828 discloses methods and compositions formicroarray controls.

In one embodiment of the invention, a nucleic acid array can comprise anarray of probes of about 15-25 nucleotides in length. In furtherembodiments, a nucleic acid array can comprise any number of probes, inwhich at least one probe is capable of detecting one or more SNPsdisclosed in Table 1 and/or Table 2, and/or at least one probe comprisesa fragment of one of the sequences selected from the group consisting ofthose disclosed in Table 1, Table 2, the Sequence Listing, and sequencescomplementary thereto, said fragment comprising at least about 8consecutive nucleotides, preferably 10, 12, 15, 16, 18, 20, morepreferably 22, 25, 30, 40, 47, 50, 55, 60, 65, 70, 80, 90, 100, or moreconsecutive nucleotides (or any other number in-between) and containing(or being complementary to) a novel SNP allele disclosed in Table 1and/or Table 2. In some embodiments, the nucleotide complementary to theSNP site is within 5, 4, 3, 2, or 1 nucleotide from the center of theprobe, more preferably at the center of said probe.

A polynucleotide probe can be synthesized on the surface of thesubstrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more polynucleotides, or any other numberwhich lends itself to the efficient use of commercially availableinstrumentation.

Using such arrays or other kits/systems, the present invention providesmethods of identifying the SNPs disclosed herein in a test sample. Suchmethods typically involve incubating a test sample of nucleic acids withan array comprising one or more probes corresponding to at least one SNPposition of the present invention, and assaying for binding of a nucleicacid from the test sample with one or more of the probes. Conditions forincubating a SNP detection reagent (or a kit/system that employs one ormore such SNP detection reagents) with a test sample vary. Incubationconditions depend on such factors as the format employed in the assay,the detection methods employed, and the type and nature of the detectionreagents used in the assay. One skilled in the art will recognize thatany one of the commonly available hybridization, amplification and arrayassay formats can readily be adapted to detect the SNPs disclosedherein.

A SNP detection kit/system of the present invention may includecomponents that are used to prepare nucleic acids from a test sample forthe subsequent amplification and/or detection of a SNP-containingnucleic acid molecule. Such sample preparation components can be used toproduce nucleic acid extracts (including DNA and/or RNA), proteins ormembrane extracts from any bodily fluids (such as blood, serum, plasma,urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin,hair, cells (especially nucleated cells), biopsies, buccal swabs ortissue specimens. The test samples used in the above-described methodswill vary based on such factors as the assay format, nature of thedetection method, and the specific tissues, cells or extracts used asthe test sample to be assayed. Methods of preparing nucleic acids,proteins, and cell extracts are well known in the art and can be readilyadapted to obtain a sample that is compatible with the system utilized.Automated sample preparation systems for extracting nucleic acids from atest sample are commercially available, and examples are Qiagen'sBioRobot 9600, Applied Biosystems' PRISM 6700, and Roche MolecularSystems' COBAS AmpliPrep System.

Another form of kit contemplated by the present invention is acompartmentalized kit. A compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers include,for example, small glass containers, plastic containers, strips ofplastic, glass or paper, or arraying material such as silica. Suchcontainers allow one to efficiently transfer reagents from onecompartment to another compartment such that the test samples andreagents are not cross-contaminated, or from one container to anothervessel not included in the kit, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother or to another vessel. Such containers may include, for example,one or more containers which will accept the test sample, one or morecontainers which contain at least one probe or other SNP detectionreagent for detecting one or more SNPs of the present invention, one ormore containers which contain wash reagents (such as phosphate bufferedsaline, Tris-buffers, etc.), and one or more containers which containthe reagents used to reveal the presence of the bound probe or other SNPdetection reagents. The kit can optionally further comprise compartmentsand/or reagents for, for example, nucleic acid amplification or otherenzymatic reactions such as primer extension reactions, hybridization,ligation, electrophoresis (preferably capillary electrophoresis), massspectrometry, and/or laser-induced fluorescent detection. The kit mayalso include instructions for using the kit. Exemplary compartmentalizedkits include microfluidic devices known in the art (see, e.g., Weigl etal., “Lab-on-a-chip for drug development”, Adv Drug Deliv Rev. 2003 Feb.24;55(3):349-77). In such microfluidic devices, the containers may bereferred to as, for example, microfluidic “compartments”, “chambers”, or“channels”.

Microfluidic devices, which may also be referred to as “lab-on-a-chip”systems, biomedical micro-electro-mechanical systems (bioMEMs), ormulticomponent integrated systems, are exemplary kits/systems of thepresent invention for analyzing SNPs. Such systems miniaturize andcompartmentalize processes such as probe/target hybridization, nucleicacid amplification, and capillary electrophoresis reactions in a singlefunctional device. Such microfluidic devices typically utilize detectionreagents in at least one aspect of the system, and such detectionreagents may be used to detect one or more SNPs of the presentinvention. One example of a microfluidic system is disclosed in U.S.Pat. No. 5,589,136, which describes the integration of PCR amplificationand capillary electrophoresis in chips. Exemplary microfluidic systemscomprise a pattern of microchannels designed onto a glass, silicon,quartz, or plastic wafer included on a microchip. The movements of thesamples may be controlled by electric, electroosmotic or hydrostaticforces applied across different areas of the microchip to createfunctional microscopic valves and pumps with no moving parts. Varyingthe voltage can be used as a means to control the liquid flow atintersections between the micro-machined channels and to change theliquid flow rate for pumping across different sections of the microchip.See, for example, U.S. Pat. No. 6,153,073, Dubrow et al., and U.S. Pat.No. 6,156,181, Parce et al.

For genotyping SNPs, an exemplary microfluidic system may integrate, forexample, nucleic acid amplification, primer extension, capillaryelectrophoresis, and a detection method such as laser inducedfluorescence detection. In a first step of an exemplary process forusing such an exemplary system, nucleic acid samples are amplified,preferably by PCR. Then, the amplification products are subjected toautomated primer extension reactions using ddNTPs (specific fluorescencefor each ddNTP) and the appropriate oligonucleotide primers to carry outprimer extension reactions which hybridize just upstream of the targetedSNP. Once the extension at the 3′ end is completed, the primers areseparated from the unincorporated fluorescent ddNTPs by capillaryelectrophoresis. The separation medium used in capillary electrophoresiscan be, for example, polyacrylamide, polyethyleneglycol or dextran. Theincorporated ddNTPs in the single nucleotide primer extension productsare identified by laser-induced fluorescence detection. Such anexemplary microchip can be used to process, for example, at least 96 to384 samples, or more, in parallel.

Uses of Nucleic Acid Molecules

The nucleic acid molecules of the present invention have a variety ofuses, especially in the diagnosis and treatment of stenosis. Forexample, the nucleic acid molecules are useful as hybridization probes,such as for genotyping SNPs in messenger RNA, transcript, cDNA, genomicDNA, amplified DNA or other nucleic acid molecules, and for isolatingfull-length cDNA and genomic clones encoding the variant peptidesdisclosed in Table 1 as well as their orthologs.

A probe can hybridize to any nucleotide sequence along the entire lengthof a nucleic acid molecule provided in Table 1 and/or Table 2.Preferably, a probe of the present invention hybridizes to a region of atarget sequence that encompasses a SNP position indicated in Table 1and/or Table 2. More preferably, a probe hybridizes to a SNP-containingtarget sequence in a sequence-specific manner such that it distinguishesthe target sequence from other nucleotide sequences which vary from thetarget sequence only by which nucleotide is present at the SNP site.Such a probe is particularly useful for detecting the presence of aSNP-containing nucleic acid in a test sample, or for determining whichnucleotide (allele) is present at a particular SNP site (i.e.,genotyping the SNP site).

A nucleic acid hybridization probe may be used for determining thepresence, level, form, and/or distribution of nucleic acid expression.The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes specific for the SNPs described herein can be usedto assess the presence, expression and/or gene copy number in a givencell, tissue, or organism. These uses are relevant for diagnosis ofdisorders involving an increase or decrease in gene expression relativeto normal levels. In vitro techniques for detection of mRNA include, forexample, Northern blot hybridizations and in situ hybridizations. Invitro techniques for detecting DNA include Southern blot hybridizationsand in situ hybridizations (Sambrook and Russell, 2000, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.).

Probes can be used as part of a diagnostic test kit for identifyingcells or tissues in which a variant protein is expressed, such as bymeasuring the level of a variant protein-encoding nucleic acid (e.g.,mRNA) in a sample of cells from a subject or determining if apolynucleotide contains a SNP of interest.

Thus, the nucleic acid molecules of the invention can be used ashybridization probes to detect the SNPs disclosed herein, therebydetermining whether an individual with the polymorphisms is at risk forstenosis or has developed early stage stenosis. Detection of a SNPassociated with a disease phenotype provides a diagnostic tool for anactive disease and/or genetic predisposition to the disease.

The nucleic acid molecules of the invention are also useful as primersto amplify any given region of a nucleic acid molecule, particularly aregion containing a SNP identified in Table 1 and/or Table 2.

The nucleic acid molecules of the invention are also useful forconstructing recombinant vectors (described in greater detail below).Such vectors include expression vectors that express a portion of, orall of, any of the variant peptide sequences provided in Table 1.Vectors also include insertion vectors, used to integrate into anothernucleic acid molecule sequence, such as into the cellular genome, toalter in situ expression of a gene and/or gene product. For example, anendogenous coding sequence can be replaced via homologous recombinationwith all or part of the coding region containing one or morespecifically introduced SNPs.

The nucleic acid molecules of the invention are also useful forexpressing antigenic portions of the variant proteins, particularlyantigenic portions that contain a variant amino acid sequence (e.g., anamino acid substitution) caused by a SNP disclosed in Table 1 and/orTable 2.

The nucleic acid molecules of the invention are also useful forconstructing vectors containing a gene regulatory region of the nucleicacid molecules of the present invention.

The nucleic acid molecules of the invention are also useful fordesigning ribozymes corresponding to all, or a part, of an mRNA moleculeexpressed from a SNP-containing nucleic acid molecule described herein.

The nucleic acid molecules of the invention are also useful forconstructing host cells expressing a part, or all, of the nucleic acidmolecules and variant peptides.

The nucleic acid molecules of the invention are also useful forconstructing transgenic animals expressing all, or a part, of thenucleic acid molecules and variant peptides. The production ofrecombinant cells and transgenic animals having nucleic acid moleculeswhich contain the SNPs disclosed in Table 1 and/or Table 2 allow, forexample, effective clinical design of treatment compounds and dosageregimens.

The nucleic acid molecules of the invention are also useful in assaysfor drug screening to identify compounds that, for example, modulatenucleic acid expression.

The nucleic acid molecules of the invention are also useful in genetherapy in patients whose cells have aberrant gene expression. Thus,recombinant cells, which include a patient's cells that have beenengineered ex vivo and returned to the patient, can be introduced intoan individual where the recombinant cells produce the desired protein totreat the individual.

SNP Genotyping Methods

The process of determining which specific nucleotide (i.e., allele) ispresent at each of one or more SNP positions, such as a SNP position ina nucleic acid molecule disclosed in Table 1 and/or Table 2, is referredto as SNP genotyping. The present invention provides methods of SNPgenotyping, such as for use in screening for stenosis or relatedpathologies, or determining predisposition thereto, or determiningresponsiveness to a form of treatment, or in genome mapping or SNPassociation analysis, etc.

Nucleic acid samples can be genotyped to determine which allele(s)is/are present at any given genetic region (e.g., SNP position) ofinterest by methods well known in the art. The neighboring sequence canbe used to design SNP detection reagents such as oligonucleotide probes,which may optionally be implemented in a kit format. Exemplary SNPgenotyping methods are described in Chen et al., “Single nucleotidepolymorphism genotyping: biochemistry, protocol, cost and throughput”,Pharmacogenomics J. 2003;3(2):77-96; Kwok et al., “Detection of singlenucleotide polymorphisms”, Curr Issues Mol. Biol. 2003 April;5(2):43-60; Shi, “Technologies for individual genotyping: detection ofgenetic polymorphisms in drug targets and disease genes”, Am JPharmacogenomics. 2002;2(3):197-205; and Kwok, “Methods for genotypingsingle nucleotide polymorphisms”, Annu Rev Genomics Hum Genet2001;2:235-58. Exemplary techniques for high-throughput SNP genotypingare described in Marnellos, “High-throughput SNP analysis for geneticassociation studies”, Curr Opin Drug Discov Devel. 2003 May;6(3):317-21. Common SNP genotyping methods include, but are not limitedto, TaqMan assays, molecular beacon assays, nucleic acid arrays,allele-specific primer extension, allele-specific PCR, arrayed primerextension, homogeneous primer extension assays, primer extension withdetection by mass spectrometry, pyrosequencing, multiplex primerextension sorted on genetic arrays, ligation with rolling circleamplification, homogeneous ligation, OLA (U.S. Pat. No. 4,988,167),multiplex ligation reaction sorted on genetic arrays,restriction-fragment length polymorphism, single base extension-tagassays, and the Invader assay. Such methods may be used in combinationwith detection mechanisms such as, for example, luminescence orchemiluminescence detection, fluorescence detection, time-resolvedfluorescence detection, fluorescence resonance energy transfer,fluorescence polarization, mass spectrometry, and electrical detection.

Various methods for detecting polymorphisms include, but are not limitedto, methods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science230:1242 (1985); Cotton et al., PNAS 85:4397 (1988); and Saleeba et al.,Meth. Enzymol. 217:286-295 (1992)), comparison of the electrophoreticmobility of variant and wild type nucleic acid molecules (Orita et al.,PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); andHayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and assayingthe movement of polymorphic or wild-type fragments in polyacrylamidegels containing a gradient of denaturant using denaturing gradient gelelectrophoresis (DGGE) (Myers et al., Nature 313:495 (1985)). Sequencevariations at specific locations can also be assessed by nucleaseprotection assays such as RNase and SI protection or chemical cleavagemethods.

In a preferred embodiment, SNP genotyping is performed using the TaqManassay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos.5,210,015 and 5,538,848). The TaqMan assay detects the accumulation of aspecific amplified product during PCR. The TaqMan assay utilizes anoligonucleotide probe labeled with a fluorescent reporter dye and aquencher dye. The reporter dye is excited by irradiation at anappropriate wavelength, it transfers energy to the quencher dye in thesame probe via a process called fluorescence resonance energy transfer(FRET). When attached to the probe, the excited reporter dye does notemit a signal. The proximity of the quencher dye to the reporter dye inthe intact probe maintains a reduced fluorescence for the reporter. Thereporter dye and quencher dye may be at the 5′ most and the 3′ mostends, respectively, or vice versa. Alternatively, the reporter dye maybe at the 5′ or 3′ most end while the quencher dye is attached to aninternal nucleotide, or vice versa. In yet another embodiment, both thereporter and the quencher may be attached to internal nucleotides at adistance from each other such that fluorescence of the reporter isreduced.

During PCR, the 5′ nuclease activity of DNA polymerase cleaves theprobe, thereby separating the reporter dye and the quencher dye andresulting in increased fluorescence of the reporter. Accumulation of PCRproduct is detected directly by monitoring the increase in fluorescenceof the reporter dye. The DNA polymerase cleaves the probe between thereporter dye and the quencher dye only if the probe hybridizes to thetarget SNP-containing template which is amplified during PCR, and theprobe is designed to hybridize to the target SNP site only if aparticular SNP allele is present.

Preferred TaqMan primer and probe sequences can readily be determinedusing the SNP and associated nucleic acid sequence information providedherein. A number of computer programs, such as Primer Express (AppliedBiosystems, Foster City, Calif.), can be used to rapidly obtain optimalprimer/probe sets. It will be apparent to one of skill in the art thatsuch primers and probes for detecting the SNPs of the present inventionare useful in diagnostic assays for stenosis and related pathologies,and can be readily incorporated into a kit format. The present inventionalso includes modifications of the Taqman assay well known in the artsuch as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and6,117,635).

Another preferred method for genotyping the SNPs of the presentinvention is the use of two oligonucleotide probes in an OLA (see, e.g.,U.S. Pat. No. 4,988,617). In this method, one probe hybridizes to asegment of a target nucleic acid with its 3′ most end aligned with theSNP site. A second probe hybridizes to an adjacent segment of the targetnucleic acid molecule directly 3′ to the first probe. The two juxtaposedprobes hybridize to the target nucleic acid molecule, and are ligated inthe presence of a linking agent such as a ligase if there is perfectcomplementarity between the 3′ most nucleotide of the first probe withthe SNP site. If there is a mismatch, ligation would not occur. Afterthe reaction, the ligated probes are separated from the target nucleicacid molecule, and detected as indicators of the presence of a SNP.

The following patents, patent applications, and published internationalpatent applications, which are all hereby incorporated by reference,provide additional information pertaining to techniques for carrying outvarious types of OLA: U.S. Pat. Nos. 6,027,889, 6,268,148, 5494810,5830711, and 6054564 describe OLA strategies for performing SNPdetection; WO 97/31256 and WO 00/56927 describe OLA strategies forperforming SNP detection using universal arrays, wherein a zipcodesequence can be introduced into one of the hybridization probes, and theresulting product, or amplified product, hybridized to a universal zipcode array; U.S. application US01/17329 (and Ser. No. 09/584,905)describes OLA (or LDR) followed by PCR, wherein zipcodes areincorporated into OLA probes, and amplified PCR products are determinedby electrophoretic or universal zipcode array readout; U.S. application60/427,818, 60/445,636, and 60/445,494 describe SNPlex methods andsoftware for multiplexed SNP detection using OLA followed by PCR,wherein zipcodes are incorporated into OLA probes, and amplified PCRproducts are hybridized with a zipchute reagent, and the identity of theSNP determined from electrophoretic readout of the zipchute. In someembodiments, OLA is carried out prior to PCR (or another method ofnucleic acid amplification). In other embodiments, PCR (or anothermethod of nucleic acid amplification) is carried out prior to OLA.

Another method for SNP genotyping is based on mass spectrometry. Massspectrometry takes advantage of the unique mass of each of the fournucleotides of DNA. SNPs can be unambiguously genotyped by massspectrometry by measuring the differences in the mass of nucleic acidshaving alternative SNP alleles. MALDI-TOF (Matrix Assisted LaserDesorption Ionization—Time of Flight) mass spectrometry technology ispreferred for extremely precise determinations of molecular mass, suchas SNPs. Numerous approaches to SNP analysis have been developed basedon mass spectrometry. Preferred mass spectrometry-based methods of SNPgenotyping include primer extension assays, which can also be utilizedin combination with other approaches, such as traditional gel-basedformats and microarrays.

Typically, the primer extension assay involves designing and annealing aprimer to a template PCR amplicon upstream (5′) from a target SNPposition. A mix of dideoxynucleotide triphosphates (ddNTPs) and/ordeoxynucleotide triphosphates (dNTPs) are added to a reaction mixturecontaining template (e.g., a SNP-containing nucleic acid molecule whichhas typically been amplified, such as by PCR), primer, and DNApolymerase. Extension of the primer terminates at the first position inthe template where a nucleotide complementary to one of the ddNTPs inthe mix occurs. The primer can be either immediately adjacent (i.e., thenucleotide at the 3′ end of the primer hybridizes to the nucleotide nextto the target SNP site) or two or more nucleotides removed from the SNPposition. If the primer is several nucleotides removed from the targetSNP position, the only limitation is that the template sequence betweenthe 3′ end of the primer and the SNP position cannot contain anucleotide of the same type as the one to be detected, or this willcause premature termination of the extension primer. Alternatively, ifall four ddNTPs alone, with no dNTPs, are added to the reaction mixture,the primer will always be extended by only one nucleotide, correspondingto the target SNP position. In this instance, primers are designed tobind one nucleotide upstream from the SNP position (i.e., the nucleotideat the 3′ end of the primer hybridizes to the nucleotide that isimmediately adjacent to the target SNP site on the 5′ side of the targetSNP site). Extension by only one nucleotide is preferable, as itminimizes the overall mass of the extended primer, thereby increasingthe resolution of mass differences between alternative SNP nucleotides.Furthermore, mass-tagged ddNTPs can be employed in the primer extensionreactions in place of unmodified ddNTPs. This increases the massdifference between primers extended with these ddNTPs, thereby providingincreased sensitivity and accuracy, and is particularly useful fortyping heterozygous base positions. Mass-tagging also alleviates theneed for intensive sample-preparation procedures and decreases thenecessary resolving power of the mass spectrometer.

The extended primers can then be purified and analyzed by MALDI-TOF massspectrometry to determine the identity of the nucleotide present at thetarget SNP position. In one method of analysis, the products from theprimer extension reaction are combined with light absorbing crystalsthat form a matrix. The matrix is then hit with an energy source such asa laser to ionize and desorb the nucleic acid molecules into thegas-phase. The ionized molecules are then ejected into a flight tube andaccelerated down the tube towards a detector. The time between theionization event, such as a laser pulse, and collision of the moleculewith the detector is the time of flight of that molecule. The time offlight is precisely correlated with the mass-to-charge ratio (m/z) ofthe ionized molecule. Ions with smaller m/z travel down the tube fasterthan ions with larger m/z and therefore the lighter ions reach thedetector before the heavier ions. The time-of-flight is then convertedinto a corresponding, and highly precise, m/z. In this manner, SNPs canbe identified based on the slight differences in mass, and thecorresponding time of flight differences, inherent in nucleic acidmolecules having different nucleotides at a single base position. Forfurther information regarding the use of primer extension assays inconjunction with MALDI-TOF mass spectrometry for SNP genotyping, see,e.g., Wise et al., “A standard protocol for single nucleotide primerextension in the human genome using matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry”, Rapid CommunMass Spectrom. 2003; 17(11):1195-202.

The following references provide further information describing massspectrometry-based methods for SNP genotyping: Bocker, “SNP and mutationdiscovery using base-specific cleavage and MALDI-TOF mass spectrometry”,Bioinformatics. 2003 July; 19 Suppl 1:144-153; Storm et al., “MALDI-TOFmass spectrometry-based SNP genotyping”, Methods Mol. Biol.2003;212:241-62; Jurinke et al., “The use of MassARRAY technology forhigh throughput genotyping”, Adv Biochem Eng Biotechnol. 2002;77:57-74;and Jurinke et al., “Automated genotyping using the DNA MassArraytechnology”, Methods Mol. Biol. 2002;187:179-92.

SNPs can also be scored by direct DNA sequencing. A variety of automatedsequencing procedures can be utilized ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO94/16101; Cohen et al., Adv. Chromatogr. 36:127-162(1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159(1993)). The nucleic acid sequences of the present invention enable oneof ordinary skill in the art to readily design sequencing primers forsuch automated sequencing procedures. Commercial instrumentation, suchas the Applied Biosystems 377, 3100, 3700, 3730, and 3730×1 DNAAnalyzers (Foster City, Calif.), is commonly used in the art forautomated sequencing.

Other methods that can be used to genotype the SNPs of the presentinvention include single-strand conformational polymorphism (SSCP), anddenaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature313:495 (1985)). SSCP identifies base differences by alteration inelectrophoretic migration of single stranded PCR products, as describedin Orita et al., Proc. Nat. Acad. Single-stranded PCR products can begenerated by heating or otherwise denaturing double stranded PCRproducts. Single-stranded nucleic acids may refold or form secondarystructures that are partially dependent on the base sequence. Thedifferent electrophoretic mobilities of single-stranded amplificationproducts are related to base-sequence differences at SNP positions. DGGEdifferentiates SNP alleles based on the different sequence-dependentstabilities and melting properties inherent in polymorphic DNA and thecorresponding differences in electrophoretic migration patterns in adenaturing gradient gel (Erlich, ed., PCR Technology, Principles andApplications for DNA Amplification, W. H. Freeman and Co, New York,1992, Chapter 7).

Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be usedto score SNPs based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. If the SNP affects a restriction enzyme cleavagesite, the SNP can be identified by alterations in restriction enzymedigestion patterns, and the corresponding changes in nucleic acidfragment lengths determined by gel electrophoresis

SNP genotyping can include the steps of, for example, collecting abiological sample from a human subject (e.g., sample of tissues, cells,fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA,mRNA or both) from the cells of the sample, contacting the nucleic acidswith one or more primers which specifically hybridize to a region of theisolated nucleic acid containing a target SNP under conditions such thathybridization and amplification of the target nucleic acid regionoccurs, and determining the nucleotide present at the SNP position ofinterest, or, in some assays, detecting the presence or absence of anamplification product (assays can be designed so that hybridizationand/or amplification will only occur if a particular SNP allele ispresent or absent). In some assays, the size of the amplificationproduct is detected and compared to the length of a control sample; forexample, deletions and insertions can be detected by a change in size ofthe amplified product compared to a normal genotype.

SNP genotyping is useful for numerous practical applications, asdescribed below. Examples of such applications include, but are notlimited to, SNP-disease association analysis, disease predispositionscreening, disease diagnosis, disease prognosis, disease progressionmonitoring, determining therapeutic strategies based on an individual'sgenotype (“pharmacogenomics”), developing therapeutic agents based onSNP genotypes associated with a disease or likelihood of responding to adrug, stratifying a patient population for clinical trial for atreatment regimen, predicting the likelihood that an individual willexperience toxic side effects from a therapeutic agent, and humanidentification applications such as forensics.

Analysis of Genetic Association Between SNPs and Phenotypic Traits

SNP genotyping for disease diagnosis, disease predisposition screening,disease prognosis, determining drug responsiveness (pharmacogenomics),drug toxicity screening, and other uses described herein, typicallyrelies on initially establishing a genetic association between one ormore specific SNPs and the particular phenotypic traits of interest.

Different study designs may be used for genetic association studies(Modern Epidemiology, Lippincott Williams & Wilkins (1998), 609-622).Observational studies are most frequently carried out in which theresponse of the patients is not interfered with. The first type ofobservational study identifies a sample of persons in whom the suspectedcause of the disease is present and another sample of persons in whomthe suspected cause is absent, and then the frequency of development ofdisease in the two samples is compared. These sampled populations arecalled cohorts, and the study is a prospective study. The other type ofobservational study is case-control or a retrospective study. In typicalcase-control studies, samples are collected from individuals with thephenotype of interest (cases) such as certain manifestations of adisease, and from individuals without the phenotype (controls) in apopulation (target population) that conclusions are to be drawn from.Then the possible causes of the disease are investigatedretrospectively. As the time and costs of collecting samples incase-control studies are considerably less than those for prospectivestudies, case-control studies are the more commonly used study design ingenetic association studies, at least during the exploration anddiscovery stage.

In both types of observational studies, there may be potentialconfounding factors that should be taken into consideration. Confoundingfactors are those that are associated with both the real cause(s) of thedisease and the disease itself, and they include demographic informationsuch as age, gender, ethnicity as well as environmental factors. Whenconfounding factors are not matched in cases and controls in a study,and are not controlled properly, spurious association results can arise.If potential confounding factors are identified, they should becontrolled for by analysis methods explained below.

In a genetic association study, the cause of interest to be tested is acertain allele or a SNP or a combination of alleles or a haplotype fromseveral SNPs. Thus, tissue specimens (e.g., whole blood) from thesampled individuals may be collected and genomic DNA genotyped for theSNP(s) of interest. In addition to the phenotypic trait of interest,other information such as demographic (e.g., age, gender, ethnicity,etc.), clinical, and environmental information that may influence theoutcome of the trait can be collected to further characterize and definethe sample set. In many cases, these factors are known to be associatedwith diseases and/or SNP allele frequencies. There are likelygene-environment and/or gene-gene interactions as well. Analysis methodsto address gene-environment and gene-gene interactions (for example, theeffects of the presence of both susceptibility alleles at two differentgenes can be greater than the effects of the individual alleles at twogenes combined) are discussed below.

After all the relevant phenotypic and genotypic information has beenobtained, statistical analyses are carried out to determine if there isany significant correlation between the presence of an allele or agenotype with the phenotypic characteristics of an individual.Preferably, data inspection and cleaning are first performed beforecarrying out statistical tests for genetic association. Epidemiologicaland clinical data of the samples can be summarized by descriptivestatistics with tables and graphs. Data validation is preferablyperformed to check for data completion, inconsistent entries, andoutliers. Chi-squared tests and t-tests (Wilcoxon rank-sum tests ifdistributions are not normal) may then be used to check for significantdifferences between cases and controls for discrete and continuousvariables, respectively. To ensure genotyping quality, Hardy-Weinbergdisequilibrium tests can be performed on cases and controls separately.Significant deviation from Hardy-Weinberg equilibrium (HWE) in bothcases and controls for individual markers can be indicative ofgenotyping errors. If HWE is violated in a majority of markers, it isindicative of population substructure that should be furtherinvestigated. Moreover, Hardy-Weinberg disequilibrium in cases only canindicate genetic association of the markers with the disease (GeneticData Analysis, Weir B., Sinauer (1990)).

To test whether an allele of a single SNP is associated with the case orcontrol status of a phenotypic trait, one skilled in the art can compareallele frequencies in cases and controls. Standard chi-squared tests andFisher exact tests can be carried out on a 2×2 table (2 SNP alleles×2outcomes in the categorical trait of interest). To test whethergenotypes of a SNP are associated, chi-squared tests can be carried outon a 3×2 table (3 genotypes×2 outcomes). Score tests are also carriedout for genotypic association to contrast the three genotypicfrequencies (major homozygotes, heterozygotes and minor homozygotes) incases and controls, and to look for trends using 3 different modes ofinheritance, namely dominant (with contrast coefficients 2, −1, −1),additive (with contrast coefficients 1, 0, −1) and recessive (withcontrast coefficients 1, 1, −2). Odds ratios for minor versus majoralleles, and odds ratios for heterozygote and homozygote variants versusthe wild type genotypes are calculated with the desired confidencelimits, usually 95%.

In order to control for confounders and to test for interaction andeffect modifiers, stratified analyses may be performed using stratifiedfactors that are likely to be confounding, including demographicinformation such as age, ethnicity, and gender, or an interactingelement or effect modifier, such as a known major gene (e.g., APOE forAlzheimer's disease or HLA genes for autoimmune diseases), orenvironmental factors such as smoking in lung cancer. Stratifiedassociation tests may be carried out using Cochran-Mantel-Haenszel teststhat take into account the ordinal nature of genotypes with 0, 1, and 2variant alleles. Exact tests by StatXact may also be performed whencomputationally possible. Another way to adjust for confounding effectsand test for interactions is to perform stepwise multiple logisticregression analysis using statistical packages such as SAS or R.Logistic regression is a model-building technique in which the bestfitting and most parsimonious model is built to describe the relationbetween the dichotomous outcome (for instance, getting a certain diseaseor not) and a set of independent variables (for instance, genotypes ofdifferent associated genes, and the associated demographic andenvironmental factors). The most common model is one in which the logittransformation of the odds ratios is expressed as a linear combinationof the variables (main effects) and their cross-product terms(interactions) (Applied Logistic Regression, Hosmer and Lemeshow, Wiley(2000)). To test whether a certain variable or interaction issignificantly associated with the outcome, coefficients in the model arefirst estimated and then tested for statistical significance of theirdeparture from zero.

In addition to performing association tests one marker at a time,haplotype association analysis may also be performed to study a numberof markers that are closely linked together. Haplotype association testscan have better power than genotypic or allelic association tests whenthe tested markers are not the disease-causing mutations themselves butare in linkage disequilibrium with such mutations. The test will even bemore powerful if the disease is indeed caused by a combination ofalleles on a haplotype (e.g., APOE is a haplotype formed by 2 SNPs thatare very close to each other). In order to perform haplotype associationeffectively, marker-marker linkage disequilibrium measures, both D′ andR², are typically calculated for the markers within a gene to elucidatethe haplotype structure. Recent studies (Daly et al, Nature Genetics,29, 232-235, 2001) in linkage disequilibrium indicate that SNPs within agene are organized in block pattern, and a high degree of linkagedisequilibrium exists within blocks and very little linkagedisequilibrium exists between blocks. Haplotype association with thedisease status can be performed using such blocks once they have beenelucidated.

Haplotype association tests can be carried out in a similar fashion asthe allelic and genotypic association tests. Each haplotype in a gene isanalogous to an allele in a multi-allelic marker. One skilled in the artcan either compare the haplotype frequencies in cases and controls ortest genetic association with different pairs of haplotypes. It has beenproposed (Schaid et al, Am. J. Hum. Genet., 70, 425-434, 2002) thatscore tests can be done on haplotypes using the program “haplo.score”.In that method, haplotypes are first inferred by EM algorithm and scoretests are carried out with a generalized linear model (GLM) frameworkthat allows the adjustment of other factors.

An important decision in the performance of genetic association tests isthe determination of the significance level at which significantassociation can be declared when the p-value of the tests reaches thatlevel. In an exploratory analysis where positive hits will be followedup in subsequent confirmatory testing, an unadjusted p-value <0.1 (asignificance level on the lenient side) may be used for generatinghypotheses for significant association of a SNP with certain phenotypiccharacteristics of a disease. It is preferred that a p-value <0.05 (asignificance level traditionally used in the art) is achieved in orderfor a SNP to be considered to have an association with a disease. It ismore preferred that a p-value <0.01 (a significance level on thestringent side) is achieved for an association to be declared. When hitsare followed up in confirmatory analyses in more samples of the samesource or in different samples from different sources, adjustment formultiple testing will be performed as to avoid excess number of hitswhile maintaining the experiment-wise error rates at 0.05. While thereare different methods to adjust for multiple testing to control fordifferent kinds of error rates, a commonly used but rather conservativemethod is Bonferroni correction to control the experiment-wise orfamily-wise error rate (Multiple comparisons and multiple tests,Westfall et al, SAS Institute (1999)). Permutation tests to control forthe false discovery rates, FDR, can be more powerful (Benjamini andHochberg, Journal of the Royal Statistical Society, Series B 57,1289-1300, 1995, Resampling-based Multiple Testing, Westfall and Young,Wiley (1993)). Such methods to control for multiplicity would bepreferred when the tests are dependent and controlling for falsediscovery rates is sufficient as opposed to controlling for theexperiment-wise error rates.

In replication studies using samples from different populations afterstatistically significant markers have been identified in theexploratory stage, meta-analyses can then be performed by combiningevidence of different studies (Modern Epidemiology, Lippincott Williams& Wilkins, 1998, 643-673). If available, association results known inthe art for the same SNPs can be included in the meta-analyses.

Since both genotyping and disease status classification can involveerrors, sensitivity analyses may be performed to see how odds ratios andp-values would change upon various estimates on genotyping and diseaseclassification error rates.

It has been well known that subpopulation-based sampling bias betweencases and controls can lead to spurious results in case-controlassociation studies (Ewens and Spielman, Am. J. Hum. Genet. 62, 450-458,1995) when prevalence of the disease is associated with differentsubpopulation groups. Such bias can also lead to a loss of statisticalpower in genetic association studies. To detect populationstratification, Pritchard and Rosenberg (Pritchard et al. Am. J. Hum.Gen. 1999, 65:220-228) suggested typing markers that are unlinked to thedisease and using results of association tests on those markers todetermine whether there is any population stratification. Whenstratification is detected, the genomic control (GC) method as proposedby Devlin and Roeder (Devlin et al. Biometrics 1999, 55:997-1004) can beused to adjust for the inflation of test statistics due to populationstratification. GC method is robust to changes in population structurelevels as well as being applicable to DNA pooling designs (Devlin et al.Genet. Epidem. 20001, 21:273-284).

While Pritchard's method recommended using 15-20 unlinked microsatellitemarkers, it suggested using more than 30 biallelic markers to get enoughpower to detect population stratification. For the GC method, it hasbeen shown (Bacanu et al. Am. J. Hum. Genet. 2000, 66:1933-1944) thatabout 60-70 biallelic markers are sufficient to estimate the inflationfactor for the test statistics due to population stratification. Hence,70 intergenic SNPs can be chosen in unlinked regions as indicated in agenome scan (Kehoe et al. Hum. Mol. Genet. 1999, 8:237-245).

Once individual risk factors, genetic or non-genetic, have been foundfor the predisposition to disease, the next step is to set up aclassification/prediction scheme to predict the category (for instance,disease or no-disease) that an individual will be in depending on hisgenotypes of associated SNPs and other non-genetic risk factors.Logistic regression for discrete trait and linear regression forcontinuous trait are standard techniques for such tasks (AppliedRegression Analysis, Draper and Smith, Wiley (1998)). Moreover, othertechniques can also be used for setting up classification. Suchtechniques include, but are not limited to, MART, CART, neural network,and discriminant analyses that are suitable for use in comparing theperformance of different methods (The Elements of Statistical Learning,Hastie, Tibshirani & Friedman, Springer (2002)).

Disease Diagnosis and Predisposition Screening

Information on association/correlation between genotypes anddisease-related phenotypes can be exploited in several ways. Forexample, in the case of a highly statistically significant associationbetween one or more SNPs with predisposition to a disease for whichtreatment is available, detection of such a genotype pattern in anindividual may justify immediate administration of treatment, or atleast the institution of regular monitoring of the individual. Detectionof the susceptibility alleles associated with serious disease in acouple contemplating having children may also be valuable to the couplein their reproductive decisions. In the case of a weaker but stillstatistically significant association between a SNP and a human disease,immediate therapeutic intervention or monitoring may not be justifiedafter detecting the susceptibility allele or SNP. Nevertheless, thesubject can be motivated to begin simple life-style changes (e.g., diet,exercise) that can be accomplished at little or no cost to theindividual but would confer potential benefits in reducing the risk ofdeveloping conditions for which that individual may have an increasedrisk by virtue of having the susceptibility allele(s).

The SNPs of the invention may contribute to stenosis in an individual indifferent ways. Some polymorphisms occur within a protein codingsequence and contribute to disease phenotype by affecting proteinstructure. Other polymorphisms occur in noncoding regions but may exertphenotypic effects indirectly via influence on, for example,replication, transcription, and/or translation. A single SNP may affectmore than one phenotypic trait. Likewise, a single phenotypic trait maybe affected by multiple SNPs in different genes.

As used herein, the terms “diagnose”, “diagnosis”, and “diagnostics”include, but are not limited to any of the following: detection ofstenosis that an individual may presently have, predisposition screening(i.e., determining the increased risk of an individual in developingstenosis in the future, or determining whether an individual has adecreased risk of developing stenosis in the future), determining aparticular type or subclass of stenosis in an individual known to havestenosis, confirming or reinforcing a previously made diagnosis ofstenosis, pharmacogenomic evaluation of an individual to determine whichtherapeutic strategy that individual is most likely to positivelyrespond to or to predict whether a patient is likely to respond to aparticular treatment, predicting whether a patient is likely toexperience toxic effects from a particular treatment or therapeuticcompound, and evaluating the future prognosis of an individual havingstenosis. Such diagnostic uses are based on the SNPs individually or ina unique combination or SNP haplotypes of the present invention.

Haplotypes are particularly useful in that, for example, fewer SNPs canbe genotyped to determine if a particular genomic region harbors a locusthat influences a particular phenotype, such as in linkagedisequilibrium-based SNP association analysis.

Linkage disequilibrium (LD) refers to the co-inheritance of alleles(e.g., alternative nucleotides) at two or more different SNP sites atfrequencies greater than would be expected from the separate frequenciesof occurrence of each allele in a given population. The expectedfrequency of co-occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in “linkage equilibrium”. In contrast, LDrefers to any non-random genetic association between allele(s) at two ormore different SNP sites, which is generally due to the physicalproximity of the two loci along a chromosome. LD can occur when two ormore SNPs sites are in close physical proximity to each other on a givenchromosome and therefore alleles at these SNP sites will tend to remainunseparated for multiple generations with the consequence that aparticular nucleotide (allele) at one SNP site will show a non-randomassociation with a particular nucleotide (allele) at a different SNPsite located nearby. Hence, genotyping one of the SNP sites will givealmost the same information as genotyping the other SNP site that is inLD.

For diagnostic purposes, if a particular SNP site is found to be usefulfor diagnosing stenosis, then the skilled artisan would recognize thatother SNP sites which are in LD with this SNP site would also be usefulfor diagnosing the condition. Various degrees of LD can be encounteredbetween two or more SNPs with the result being that some SNPs are moreclosely associated (i.e., in stronger LD) than others. Furthermore, thephysical distance over which LD extends along a chromosome differsbetween different regions of the genome, and therefore the degree ofphysical separation between two or more SNP sites necessary for LD tooccur can differ between different regions of the genome.

For diagnostic applications, polymorphisms (e.g., SNPs and/orhaplotypes) that are not the actual disease-causing (causative)polymorphisms, but are in LD with such causative polymorphisms, are alsouseful. In such instances, the genotype of the polymorphism(s) thatis/are in LD with the causative polymorphism is predictive of thegenotype of the causative polymorphism and, consequently, predictive ofthe phenotype (e.g., stenosis) that is influenced by the causativeSNP(s). Thus, polymorphic markers that are in LD with causativepolymorphisms are useful as diagnostic markers, and are particularlyuseful when the actual causative polymorphism(s) is/are unknown.

Linkage disequilibrium in the human genome is reviewed in: Wall et al.,“Haplotype blocks and linkage disequilibrium in the human genome”, NatRev Genet. 2003 August; 4(8):587-97; Gamer et al., “On selecting markersfor association studies: patterns of linkage disequilibrium between twoand three diallelic loci”, Genet Epidemiol. 2003 January; 24(1):57-67;Ardlie et al., “Patterns of linkage disequilibrium in the human genome”,Nat Rev Genet. 2002 April; 3(4):299-309 (erratum in Nat Rev Genet 2002July; 3(7):566); and Remm et al., “High-density genotyping and linkagedisequilibrium in the human genome using chromosome 22 as a model”; CurrOpin Chem Biol. 2002 February; 6(1):24-30.

The contribution or association of particular SNPs and/or SNP haplotypeswith disease phenotypes, such as stenosis, enables the SNPs of thepresent invention to be used to develop superior diagnostic testscapable of identifying individuals who express a detectable trait, suchas stenosis, as the result of a specific genotype, or individuals whosegenotype places them at an increased or decreased risk of developing adetectable trait at a subsequent time as compared to individuals who donot have that genotype. As described herein, diagnostics may be based ona single SNP or a group of SNPs. Combined detection of a plurality ofSNPs (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 24, 25, 30, 32, 48, 50, 64, 96, 100, or any other numberin-between, or more, of the SNPs provided in Table 1 and/or Table 2)typically increases the probability of an accurate diagnosis. Forexample, the presence of a single SNP known to correlate with stenosismight indicate a probability of 20% that an individual has or is at riskof developing stenosis, whereas detection of five SNPs, each of whichcorrelates with stenosis, might indicate a probability of 80% that anindividual has or is at risk of developing stenosis. To further increasethe accuracy of diagnosis or predisposition screening, analysis of theSNPs of the present invention can be combined with that of otherpolymorphisms or other risk factors of stenosis, such as diseasesymptoms, pathological characteristics, family history, diet,environmental factors or lifestyle factors.

It will, of course, be understood by practitioners skilled in thetreatment or diagnosis of stenosis that the present invention generallydoes not intend to provide an absolute identification of individuals whoare at risk (or less at risk) of developing stenosis, and/or pathologiesrelated to stenosis, but rather to indicate a certain increased (ordecreased) degree or likelihood of developing the disease based onstatistically significant association results. However, this informationis extremely valuable as it can be used to, for example, initiatepreventive treatments or to allow an individual carrying one or moresignificant SNPs or SNP haplotypes to foresee warning signs such asminor clinical symptoms, or to have regularly scheduled physical examsto monitor for appearance of a condition in order to identify and begintreatment of the condition at an early stage. Particularly with diseasesthat are extremely debilitating or fatal if not treated on time, theknowledge of a potential predisposition, even if this predisposition isnot absolute, would likely contribute in a very significant manner totreatment efficacy.

The diagnostic techniques of the present invention may employ a varietyof methodologies to determine whether a test subject has a SNP or a SNPpattern associated with an increased or decreased risk of developing adetectable trait or whether the individual suffers from a detectabletrait as a result of a particular polymorphism/mutation, including, forexample, methods which enable the analysis of individual chromosomes forhaplotyping, family studies, single sperm DNA analysis, or somatichybrids. The trait analyzed using the diagnostics of the invention maybe any detectable trait that is commonly observed in pathologies anddisorders related to stenosis.

Another aspect of the present invention relates to a method ofdetermining whether an individual is at risk (or less at risk) ofdeveloping one or more traits or whether an individual expresses one ormore traits as a consequence of possessing a particular trait-causing ortrait-influencing allele. These methods generally involve obtaining anucleic acid sample from an individual and assaying the nucleic acidsample to determine which nucleotide(s) is/are present at one or moreSNP positions, wherein the assayed nucleotide(s) is/are indicative of anincreased or decreased risk of developing the trait or indicative thatthe individual expresses the trait as a result of possessing aparticular trait-causing or trait-influencing allele.

In another embodiment, the SNP detection reagents of the presentinvention are used to determine whether an individual has one or moreSNP allele(s) affecting the level (e.g., the concentration of mRNA orprotein in a sample, etc.) or pattern (e.g., the kinetics of expression,rate of decomposition, stability profile, Km, Vmax, etc.) of geneexpression (collectively, the “gene response” of a cell or bodilyfluid). Such a determination can be accomplished by screening for mRNAor protein expression (e.g., by using nucleic acid arrays, RT-PCR,TaqMan assays, or mass spectrometry), identifying genes having alteredexpression in an individual, genotyping SNPs disclosed in Table 1 and/orTable 2 that could affect the expression of the genes having alteredexpression (e.g., SNPs that are in and/or around the gene(s) havingaltered expression, SNPs in regulatory/control regions, SNPs in and/oraround other genes that are involved in pathways that could affect theexpression of the gene(s) having altered expression, or all SNPs couldbe genotyped), and correlating SNP genotypes with altered geneexpression. In this manner, specific SNP alleles at particular SNP sitescan be identified that affect gene expression.

Pharmacogenomics and Therapeutics/Drug Development

The present invention provides methods for assessing thepharmacogenomics of a subject harboring particular SNP alleles orhaplotypes to a particular therapeutic agent or pharmaceutical compound,or to a class of such compounds. Pharmacogenomics deals with the roleswhich clinically significant hereditary variations (e.g., SNPs) play inthe response to drugs due to altered drug disposition and/or abnormalaction in affected persons. See, e.g., Roses, Nature 405, 857-865(2000); Gould Rothberg, Nature Biotechnology 19, 209-211 (2001);Eichelbaum, Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996); andLinder, Clin. Chem. 43(2):254-266 (1997). The clinical outcomes of thesevariations can result in severe toxicity of therapeutic drugs in certainindividuals or therapeutic failure of drugs in certain individuals as aresult of individual variation in metabolism. Thus, the SNP genotype ofan individual can determine the way a therapeutic compound acts on thebody or the way the body metabolizes the compound. For example, SNPs indrug metabolizing enzymes can affect the activity of these enzymes,which in turn can affect both the intensity and duration of drug action,as well as drug metabolism and clearance.

The discovery of SNPs in drug metabolizing enzymes, drug transporters,proteins for pharmaceutical agents, and other drug targets has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. SNPs can be expressed in the phenotype of the extensivemetabolizer and in the phenotype of the poor metabolizer. Accordingly,SNPs may lead to allelic variants of a protein in which one or more ofthe protein functions in one population are different from those inanother population. SNPs and the encoded variant peptides thus providetargets to ascertain a genetic predisposition that can affect treatmentmodality. For example, in a ligand-based treatment, SNPs may give riseto amino terminal extracellular domains and/or other ligand-bindingregions of a receptor that are more or less active in ligand binding,thereby affecting subsequent protein activation. Accordingly, liganddosage would necessarily be modified to maximize the therapeutic effectwithin a given population containing particular SNP alleles orhaplotypes.

As an alternative to genotyping, specific variant proteins containingvariant amino acid sequences encoded by alternative SNP alleles could beidentified. Thus, pharmacogenomic characterization of an individualpermits the selection of effective compounds and effective dosages ofsuch compounds for prophylactic or therapeutic uses based on theindividual's SNP genotype, thereby enhancing and optimizing theeffectiveness of the therapy. Furthermore, the production of recombinantcells and transgenic animals containing particular SNPs/haplotypes alloweffective clinical design and testing of treatment compounds and dosageregimens. For example, transgenic animals can be produced that differonly in specific SNP alleles in a gene that is orthologous to a humandisease susceptibility gene.

Pharmacogenomic uses of the SNPs of the present invention provideseveral significant advantages for patient care, particularly intreating stenosis. Pharmacogenomic characterization of an individual,based on an individual's SNP genotype, can identify those individualsunlikely to respond to treatment with a particular medication andthereby allows physicians to avoid prescribing the ineffectivemedication to those individuals. On the other hand, SNP genotyping of anindividual may enable physicians to select the appropriate medicationand dosage regimen that will be most effective based on an individual'sSNP genotype. This information increases a physician's confidence inprescribing medications and motivates patients to comply with their drugregimens. Furthermore, pharmacogenomics may identify patientspredisposed to toxicity and adverse reactions to particular drugs ordrug dosages. Adverse drug reactions lead to more than 100,000 avoidabledeaths per year in the United States alone and therefore represent asignificant cause of hospitalization and death, as well as a significanteconomic burden on the healthcare system (Pfost et. al., Trends inBiotechnology, August 2000.). Thus, pharmacogenomics based on the SNPsdisclosed herein has the potential to both save lives and reducehealthcare costs substantially.

Pharmacogenomics in general is discussed further in Rose et al.,“Pharmacogenetic analysis of clinically relevant genetic polymorphisms”,Methods Mol Med. 2003;85:225-37. Pharmacogenomics as it relates toAlzheimer's disease and other neurodegenerative disorders is discussedin Cacabelos, “Pharmacogenomics for the treatment of dementia”, Ann Med.2002;34(5):357-79, Maimone et al., “Pharmacogenomics ofneurodegenerative diseases”, Eur J Pharmacol. 2001 Feb. 9;413(1):11-29,and Poirier, “Apolipoprotein E: a pharmacogenetic target for thetreatment of Alzheimer's disease”, Mol Diagn. 1999 December;4(4):335-41. Pharmacogenomics as it relates to cardiovascular disordersis discussed in Siest et al., “Pharmacogenomics of drugs affecting thecardiovascular system”, Clin Chem Lab Med. 2003 April; 41(4):590-9,Mukherjee et al., “Pharmacogenomics in cardiovascular diseases”, ProgCardiovasc Dis. 2002 May-June; 44(6):479-98, and Mooser et al.,“Cardiovascular pharmacogenetics in the SNP era”, J Thromb Haemost. 2003July; 1(7):1398-402. Pharmacogenomics as it relates to cancer isdiscussed in McLeod et al., “Cancer pharmacogenomics: SNPs, chips, andthe individual patient”, Cancer Invest. 2003;21(4):630-40 and Watters etal., “Cancer pharmacogenomics: current and future applications”, BiochimBiophys Acta. 2003 Mar. 17; 1603(2):99-111.

The SNPs of the present invention also can be used to identify noveltherapeutic targets for stenosis. For example, genes containing thedisease-associated variants (“variant genes”) or their products, as wellas genes or their products that are directly or indirectly regulated byor interacting with these variant genes or their products, can betargeted for the development of therapeutics that, for example, treatthe disease or prevent or delay disease onset. The therapeutics may becomposed of, for example, small molecules, proteins, protein fragmentsor peptides, antibodies, nucleic acids, or their derivatives or mimeticswhich modulate the functions or levels of the target genes or geneproducts.

The SNP-containing nucleic acid molecules disclosed herein, and theircomplementary nucleic acid molecules, may be used as antisenseconstructs to control gene expression in cells, tissues, and organisms.Antisense technology is well established in the art and extensivelyreviewed in Antisense Drug Technology: Principles, Strategies, andApplications, Crooke (ed.), Marcel Dekker, Inc.: New York (2001). Anantisense nucleic acid molecule is generally designed to becomplementary to a region of mRNA expressed by a gene so that theantisense molecule hybridizes to the mRNA and thereby blocks translationof mRNA into protein. Various classes of antisense oligonucleotides areused in the art, two of which are cleavers and blockers. Cleavers, bybinding to target RNAs, activate intracellular nucleases (e.g., RNaseHor RNase L) that cleave the target RNA. Blockers, which also bind totarget RNAs, inhibit protein translation through steric hindrance ofribosomes. Exemplary blockers include peptide nucleic acids,morpholinos, locked nucleic acids, and methylphosphonates (see, e.g.,Thompson, Drug Discovery Today, 7 (17): 912-917 (2002)). Antisenseoligonucleotides are directly useful as therapeutic agents, and are alsouseful for determining and validating gene function (e.g., in geneknock-out or knock-down experiments).

Antisense technology is further reviewed in: Layery et al., “Antisenseand RNAi: powerful tools in drug target discovery and validation”, CurrOpin Drug Discov Devel. 2003 July; 6(4):561-9; Stephens et al.,“Antisense oligonucleotide therapy in cancer”, Curr Opin Mol Ther. 2003April; 5(2): 118-22; Kurreck, “Antisense technologies. Improvementthrough novel chemical modifications”, Eur J. Biochem. 2003 April;270(8):1628-44; Dias et al., “Antisense oligonucleotides: basic conceptsand mechanisms”, Mol Cancer Ther. 2002 March; 1(5):347-55; Chen,“Clinical development of antisense oligonucleotides as anti-cancertherapeutics”, Methods Mol Med. 2003;75:621-36; Wang et al., “Antisenseanticancer oligonucleotide therapeutics”, Curr Cancer Drug Targets. 2001November; 1 (3):177-96; and Bennett, “Efficiency of antisenseoligonucleotide drug discovery”, Antisense Nucleic Acid Drug Dev. 2002June; 12(3):215-24.

The SNPs of the present invention are particularly useful for designingantisense reagents that are specific for particular nucleic acidvariants. Based on the SNP information disclosed herein, antisenseoligonucleotides can be produced that specifically target mRNA moleculesthat contain one or more particular SNP nucleotides. In this manner,expression of mRNA molecules that contain one or more undesiredpolymorphisms (e.g., SNP nucleotides that lead to a defective proteinsuch as an amino acid substitution in a catalytic domain) can beinhibited or completely blocked. Thus, antisense oligonucleotides can beused to specifically bind a particular polymorphic form (e.g., a SNPallele that encodes a defective protein), thereby inhibiting translationof this form, but which do not bind an alternative polymorphic form(e.g., an alternative SNP nucleotide that encodes a protein havingnormal function).

Antisense molecules can be used to inactivate mRNA in order to inhibitgene expression and production of defective proteins. Accordingly, thesemolecules can be used to treat a disorder, such as stenosis,characterized by abnormal or undesired gene expression or expression ofcertain defective proteins. This technique can involve cleavage by meansof ribozymes containing nucleotide sequences complementary to one ormore regions in the mRNA that attenuate the ability of the mRNA to betranslated. Possible mRNA regions include, for example, protein-codingregions and particularly protein-coding regions corresponding tocatalytic activities, substrate/ligand binding, or other functionalactivities of a protein.

The SNPs of the present invention are also useful for designing RNAinterference reagents that specifically target nucleic acid moleculeshaving particular SNP variants. RNA interference (RNAi), also referredto as gene silencing, is based on using double-stranded RNA (dsRNA)molecules to turn genes off. When introduced into a cell, dsRNAs areprocessed by the cell into short fragments (generally about 21-22 bp inlength) known as small interfering RNAs (siRNAs) which the cell uses ina sequence-specific manner to recognize and destroy complementary RNAs(Thompson, Drug Discovery Today, 7 (17): 912-917 (2002)). Thus, becauseRNAi molecules, including siRNAs, act in a sequence-specific manner, theSNPs of the present invention can be used to design RNAi reagents thatrecognize and destroy nucleic acid molecules having specific SNPalleles/nucleotides (such as deleterious alleles that lead to theproduction of defective proteins), while not affecting nucleic acidmolecules having alternative SNP alleles (such as alleles that encodeproteins having normal function). As with antisense reagents, RNAireagents may be directly useful as therapeutic agents (e.g., for turningoff defective, disease-causing genes), and are also useful forcharacterizing and validating gene function (e.g., in gene knock-out orknock-down experiments).

The following references provide a further review of RNAi: Agami, “RNAiand related mechanisms and their potential use for therapy”, Curr OpinChem Biol. 2002 December; 6(6):829-34; Layery et al., “Antisense andRNAi: powerful tools in drug target discovery and validation”, Curr OpinDrug Discov Devel. 2003 July; 6(4):561-9; Shi, “Mammalian RNAi for themasses”, Trends Genet 2003 January; 19(1):9-12), Shuey et al., “RNAi:gene-silencing in therapeutic intervention”, Drug Discovery Today 2002October; 7(20):1040-1046; McManus et al., Nat Rev Genet 2002 October;3(10):737-47; Xia et al., Nat Biotechnol 2002 October; 20(10):1006-10;Plasterk et al., Curr Opin Genet Dev 2000 October; 10(5):562-7; Bosheret al., Nat Cell Biol 2000 February; 2(2):E31-6; and Hunter, Curr Biol1999 Jun. 17;9(12):R440-2).

A subject suffering from a pathological condition, such as stenosis,ascribed to a SNP may be treated so as to correct the genetic defect(see Kren et al., Proc. Natl. Acad. Sci. USA 96:10349-10354 (1999)).Such a subject can be identified by any method that can detect thepolymorphism in a biological sample drawn from the subject. Such agenetic defect may be permanently corrected by administering to such asubject a nucleic acid fragment incorporating a repair sequence thatsupplies the normal/wild-type nucleotide at the position of the SNP.This site-specific repair sequence can encompass an RNA/DNAoligonucleotide that operates to promote endogenous repair of asubject's genomic DNA. The site-specific repair sequence is administeredin an appropriate vehicle, such as a complex with polyethylenimine,encapsulated in anionic liposomes, a viral vector such as an adenovirus,or other pharmaceutical composition that promotes intracellular uptakeof the administered nucleic acid. A genetic defect leading to an inbornpathology may then be overcome, as the chimeric oligonucleotides induceincorporation of the normal sequence into the subject's genome. Uponincorporation, the normal gene product is expressed, and the replacementis propagated, thereby engendering a permanent repair and therapeuticenhancement of the clinical condition of the subject.

In cases in which a cSNP results in a variant protein that is ascribedto be the cause of, or a contributing factor to, a pathologicalcondition, a method of treating such a condition can includeadministering to a subject experiencing the pathology thewild-type/normal cognate of the variant protein. Once administered in aneffective dosing regimen, the wild-type cognate provides complementationor remediation of the pathological condition.

The invention further provides a method for identifying a compound oragent that can be used to treat stenosis. The SNPs disclosed herein areuseful as targets for the identification and/or development oftherapeutic agents. A method for identifying a therapeutic agent orcompound typically includes assaying the ability of the agent orcompound to modulate the activity and/or expression of a SNP-containingnucleic acid or the encoded product and thus identifying an agent or acompound that can be used to treat a disorder characterized by undesiredactivity or expression of the SNP-containing nucleic acid or the encodedproduct. The assays can be performed in cell-based and cell-freesystems. Cell-based assays can include cells naturally expressing thenucleic acid molecules of interest or recombinant cells geneticallyengineered to express certain nucleic acid molecules.

Variant gene expression in a stenosis patient can include, for example,either expression of a SNP-containing nucleic acid sequence (forinstance, a gene that contains a SNP can be transcribed into an mRNAtranscript molecule containing the SNP, which can in turn be translatedinto a variant protein) or altered expression of a normal/wild-typenucleic acid sequence due to one or more SNPs (for instance, aregulatory/control region can contain a SNP that affects the level orpattern of expression of a normal transcript).

Assays for variant gene expression can involve direct assays of nucleicacid levels (e.g., mRNA levels), expressed protein levels, or ofcollateral compounds involved in a signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thesignal pathway can also be assayed. In this embodiment, the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

Modulators of variant gene expression can be identified in a methodwherein, for example, a cell is contacted with a candidatecompound/agent and the expression of mRNA determined. The level ofexpression of mRNA in the presence of the candidate compound is comparedto the level of expression of mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof variant gene expression based on this comparison and be used to treata disorder such as stenosis that is characterized by variant geneexpression (e.g., either expression of a SNP-containing nucleic acid oraltered expression of a normal/wild-type nucleic acid molecule due toone or more SNPs that affect expression of the nucleic acid molecule)due to one or more SNPs of the present invention. When expression ofmRNA is statistically significantly greater in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thecandidate compound than in its absence, the candidate compound isidentified as an inhibitor of nucleic acid expression.

The invention further provides methods of treatment, with the SNP orassociated nucleic acid domain (e.g., catalytic domain,ligand/substrate-binding domain, regulatory/control region, etc.) orgene, or the encoded mRNA transcript, as a target, using a compoundidentified through drug screening as a gene modulator to modulatevariant nucleic acid expression. Modulation can include eitherup-regulation (i.e., activation or agonization) or down-regulation(i.e., suppression or antagonization) of nucleic acid expression.

Expression of mRNA transcripts and encoded proteins, either wild type orvariant, may be altered in individuals with a particular SNP allele in aregulatory/control element, such as a promoter or transcription factorbinding domain, that regulates expression. In this situation, methods oftreatment and compounds can be identified, as discussed herein, thatregulate or overcome the variant regulatory/control element, therebygenerating normal, or healthy, expression levels of either the wild typeor variant protein.

The SNP-containing nucleic acid molecules of the present invention arealso useful for monitoring the effectiveness of modulating compounds onthe expression or activity of a variant gene, or encoded product, inclinical trials or in a treatment regimen. Thus, the gene expressionpattern can serve as an indicator for the continuing effectiveness oftreatment with the compound, particularly with compounds to which apatient can develop resistance, as well as an indicator for toxicities.The gene expression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

In another aspect of the present invention, there is provided apharmaceutical pack comprising a therapeutic agent (e.g., a smallmolecule drug, antibody, peptide, antisense or RNAi nucleic acidmolecule, etc.) and a set of instructions for administration of thetherapeutic agent to humans diagnostically tested for one or more SNPsor SNP haplotypes provided by the present invention.

The SNPs/haplotypes of the present invention are also useful forimproving many different aspects of the drug development process. Forexample, individuals can be selected for clinical trials based on theirSNP genotype. Individuals with SNP genotypes that indicate that they aremost likely to respond to the drug can be included in the trials andthose individuals whose SNP genotypes indicate that they are less likelyto or would not respond to the drug, or suffer adverse reactions, can beeliminated from the clinical trials. This not only improves the safetyof clinical trials, but also will enhance the chances that the trialwill demonstrate statistically significant efficacy. Furthermore, theSNPs of the present invention may explain why certain previouslydeveloped drugs performed poorly in clinical trials and may helpidentify a subset of the population that would benefit from a drug thathad previously performed poorly in clinical trials, thereby “rescuing”previously developed drugs, and enabling the drug to be made availableto a particular stenosis patient population that can benefit from it.

SNPs have many important uses in drug discovery, screening, anddevelopment. A high probability exists that, for any gene/proteinselected as a potential drug target, variants of that gene/protein willexist in a patient population. Thus, determining the impact ofgene/protein variants on the selection and delivery of a therapeuticagent should be an integral aspect of the drug discovery and developmentprocess. (Jazwinska, A Trends Guide to Genetic Variation and GenomicMedicine, 2002 March; S30-S36).

Knowledge of variants (e.g., SNPs and any corresponding amino acidpolymorphisms) of a particular therapeutic target (e.g., a gene, mRNAtranscript, or protein) enables parallel screening of the variants inorder to identify therapeutic candidates (e.g., small moleculecompounds, antibodies, antisense or RNAi nucleic acid compounds, etc.)that demonstrate efficacy across variants (Rothberg, Nat Biotechnol 2001March; 19(3):209-11). Such therapeutic candidates would be expected toshow equal efficacy across a larger segment of the patient population,thereby leading to a larger potential market for the therapeuticcandidate.

Furthermore, identifying variants of a potential therapeutic targetenables the most common form of the target to be used for selection oftherapeutic candidates, thereby helping to ensure that the experimentalactivity that is observed for the selected candidates reflects the realactivity expected in the largest proportion of a patient population(Jazwinska, A Trends Guide to Genetic Variation and Genomic Medicine,2002 March; S30-S36).

Additionally, screening therapeutic candidates against all knownvariants of a target can enable the early identification of potentialtoxicities and adverse reactions relating to particular variants. Forexample, variability in drug absorption, distribution, metabolism andexcretion (ADME) caused by, for example, SNPs in therapeutic targets ordrug metabolizing genes, can be identified, and this information can beutilized during the drug development process to minimize variability indrug disposition and develop therapeutic agents that are safer across awider range of a patient population. The SNPs of the present invention,including the variant proteins and encoding polymorphic nucleic acidmolecules provided in Tables 1-2, are useful in conjunction with avariety of toxicology methods established in the art, such as those setforth in Current Protocols in Toxicology, John Wiley & Sons, Inc., N.Y.

Furthermore, therapeutic agents that target any art-known proteins (ornucleic acid molecules, either RNA or DNA) may cross-react with thevariant proteins (or polymorphic nucleic acid molecules) disclosed inTable 1, thereby significantly affecting the pharmacokinetic propertiesof the drug. Consequently, the protein variants and the SNP-containingnucleic acid molecules disclosed in Tables 1-2 are useful in developing,screening, and evaluating therapeutic agents that target correspondingart-known protein forms (or nucleic acid molecules). Additionally, asdiscussed above, knowledge of all polymorphic forms of a particular drugtarget enables the design of therapeutic agents that are effectiveagainst most or all such polymorphic forms of the drug target.

Pharmaceutical Compositions and Administration Thereof

Any of the stenosis-associated proteins, and encoding nucleic acidmolecules, disclosed herein can be used as therapeutic targets (ordirectly used themselves as therapeutic compounds) for treating stenosisand related pathologies, and the present disclosure enables therapeuticcompounds (e.g., small molecules, antibodies, therapeutic proteins, RNAiand antisense molecules, etc.) to be developed that target (or arecomprised of) any of these therapeutic targets.

In general, a therapeutic compound will be administered in atherapeutically effective amount by any of the accepted modes ofadministration for agents that serve similar utilities. The actualamount of the therapeutic compound of this invention, i.e., the activeingredient, will depend upon numerous factors such as the severity ofthe disease to be treated, the age and relative health of the subject,the potency of the compound used, the route and form of administration,and other factors.

Therapeutically effective amounts of therapeutic compounds may rangefrom, for example, approximately 0.01-50 mg per kilogram body weight ofthe recipient per day; preferably about 0.1-20 mg/kg/day. Thus, as anexample, for administration to a 70 kg person, the dosage range wouldmost preferably be about 7 mg to 1.4 g per day.

In general, therapeutic compounds will be administered as pharmaceuticalcompositions by any one of the following routes: oral, systemic (e.g.,transdermal, intranasal, or by suppository), or parenteral (e.g.,intramuscular, intravenous, or subcutaneous) administration. Thepreferred manner of administration is oral or parenteral using aconvenient daily dosage regimen, which can be adjusted according to thedegree of affliction. Oral compositions can take the form of tablets,pills, capsules, semisolids, powders, sustained release formulations,solutions, suspensions, elixirs, aerosols, or any other appropriatecompositions.

The choice of formulation depends on various factors such as the mode ofdrug administration (e.g., for oral administration, formulations in theform of tablets, pills, or capsules are preferred) and thebioavailability of the drug substance. Recently, pharmaceuticalformulations have been developed especially for drugs that show poorbioavailability based upon the principle that bioavailability can beincreased by increasing the surface area, i.e., decreasing particlesize. For example, U.S. Pat. No. 4,107,288 describes a pharmaceuticalformulation having particles in the size range from 10 to 1,000 nm inwhich the active material is supported on a cross-linked matrix ofmacromolecules. U.S. Pat. No. 5,145,684 describes the production of apharmaceutical formulation in which the drug substance is pulverized tonanoparticles (average particle size of 400 nm) in the presence of asurface modifier and then dispersed in a liquid medium to give apharmaceutical formulation that exhibits remarkably highbioavailability.

Pharmaceutical compositions are comprised of, in general, a therapeuticcompound in combination with at least one pharmaceutically acceptableexcipient. Acceptable excipients are non-toxic, aid administration, anddo not adversely affect the therapeutic benefit of the therapeuticcompound. Such excipients may be any solid, liquid, semi-solid or, inthe case of an aerosol composition, gaseous excipient that is generallyavailable to one skilled in the art.

Solid pharmaceutical excipients include starch, cellulose, talc,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, magnesium stearate, sodium stearate, glycerol monostearate, sodiumchloride, dried skim milk and the like. Liquid and semisolid excipientsmay be selected from glycerol, propylene glycol, water, ethanol andvarious oils, including those of petroleum, animal, vegetable orsynthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesameoil, etc. Preferred liquid carriers, particularly for injectablesolutions, include water, saline, aqueous dextrose, and glycols.

Compressed gases may be used to disperse a compound of this invention inaerosol form. Inert gases suitable for this purpose are nitrogen, carbondioxide, etc.

Other suitable pharmaceutical excipients and their formulations aredescribed in Remington's Pharmaceutical Sciences, edited by E. W. Martin(Mack Publishing Company, 18th ed., 1990).

The amount of the therapeutic compound in a formulation can vary withinthe full range employed by those skilled in the art. Typically, theformulation will contain, on a weight percent (wt %) basis, from about0.01-99.99 wt % of the therapeutic compound based on the totalformulation, with the balance being one or more suitable pharmaceuticalexcipients. Preferably, the compound is present at a level of about 1-80wt %.

Therapeutic compounds can be administered alone or in combination withother therapeutic compounds or in combination with one or more otheractive ingredient(s). For example, an inhibitor or stimulator of astenosis-associated protein can be administered in combination withanother agent that inhibits or stimulates the activity of the same or adifferent stenosis-associated protein to thereby counteract the affectsof stenosis.

For further information regarding pharmacology, see Current Protocols inPharmacology, John Wiley & Sons, Inc., N.Y.

Human Identification Applications

In addition to their diagnostic and therapeutic uses in stenosis andrelated pathologies, the SNPs provided by the present invention are alsouseful as human identification markers for such applications asforensics, paternity testing, and biometrics (see, e.g., Gill, “Anassessment of the utility of single nucleotide polymorphisms (SNPs) forforensic purposes”, Int J Legal Med. 2001;114(4-5):204-10). Geneticvariations in the nucleic acid sequences between individuals can be usedas genetic markers to identify individuals and to associate a biologicalsample with an individual. Determination of which nucleotides occupy aset of SNP positions in an individual identifies a set of SNP markersthat distinguishes the individual. The more SNP positions that areanalyzed, the lower the probability that the set of SNPs in oneindividual is the same as that in an unrelated individual. Preferably,if multiple sites are analyzed, the sites are unlinked (i.e., inheritedindependently). Thus, preferred sets of SNPs can be selected from amongthe SNPs disclosed herein, which may include SNPs on differentchromosomes, SNPs on different chromosome arms, and/or SNPs that aredispersed over substantial distances along the same chromosome arm.

Furthermore, among the SNPs disclosed herein, preferred SNPs for use incertain forensic/human identification applications include SNPs locatedat degenerate codon positions (i.e., the third position in certaincodons which can be one of two or more alternative nucleotides and stillencode the same amino acid), since these SNPs do not affect the encodedprotein. SNPs that do not affect the encoded protein are expected to beunder less selective pressure and are therefore expected to be morepolymorphic in a population, which is typically an advantage forforensic/human identification applications. However, for certainforensics/human identification applications, such as predictingphenotypic characteristics (e.g., inferring ancestry or inferring one ormore physical characteristics of an individual) from a DNA sample, itmay be desirable to utilize SNPs that affect the encoded protein.

For many of the SNPs disclosed in Tables 1-2 (which are identified as“Applera” SNP source), Tables 1-2 provide SNP allele frequenciesobtained by re-sequencing the DNA of chromosomes from 39 individuals(Tables 1-2 also provide allele frequency information for “Celera”source SNPs and, where available, public SNPs from dbEST, HGBASE, and/orHGMD). The allele frequencies provided in Tables 1-2 enable these SNPsto be readily used for human identification applications. Although anySNP disclosed in Table 1 and/or Table 2 could be used for humanidentification, the closer that the frequency of the minor allele at aparticular SNP site is to 50%, the greater the ability of that SNP todiscriminate between different individuals in a population since itbecomes increasingly likely that two randomly selected individuals wouldhave different alleles at that SNP site. Using the SNP allelefrequencies provided in Tables 1-2, one of ordinary skill in the artcould readily select a subset of SNPs for which the frequency of theminor allele is, for example, at least 1%, 2%, 5%, 10%, 20%, 25%, 30%,40%, 45%, or 50%, or any other frequency in-between. Thus, since Tables1-2 provide allele frequencies based on the re-sequencing of thechromosomes from 39 individuals, a subset of SNPs could readily beselected for human identification in which the total allele count of theminor allele at a particular SNP site is, for example, at least 1, 2, 4,8, 10, 16, 20, 24, 30, 32, 36, 38, 39, 40, or any other numberin-between.

Furthermore, Tables 1-2 also provide population group (interchangeablyreferred to herein as ethnic or racial groups) information coupled withthe extensive allele frequency information. For example, the group of 39individuals whose DNA was re-sequenced was made-up of 20 Caucasians and19 African-Americans. This population group information enables furtherrefinement of SNP selection for human identification. For example,preferred SNPs for human identification can be selected from Tables 1-2that have similar allele frequencies in both the Caucasian andAfrican-American populations; thus, for example, SNPs can be selectedthat have equally high discriminatory power in both populations.Alternatively, SNPs can be selected for which there is a statisticallysignificant difference in allele frequencies between the Caucasian andAfrican-American populations (as an extreme example, a particular allelemay be observed only in either the Caucasian or the African-Americanpopulation group but not observed in the other population group); suchSNPs are useful, for example, for predicting the race/ethnicity of anunknown perpetrator from a biological sample such as a hair or bloodstain recovered at a crime scene. For a discussion of using SNPs topredict ancestry from a DNA sample, including statistical methods, seeFrudakis et al., “A Classifier for the SNP-Based Inference of Ancestry”,Journal of Forensic Sciences 2003; 48(4):771-782.

SNPs have numerous advantages over other types of polymorphic markers,such as short tandem repeats (STRs). For example, SNPs can be easilyscored and are amenable to automation, making SNPs the markers of choicefor large-scale forensic databases. SNPs are found in much greaterabundance throughout the genome than repeat polymorphisms. Populationfrequencies of two polymorphic forms can usually be determined withgreater accuracy than those of multiple polymorphic forms atmulti-allelic loci. SNPs are mutationaly more stable than repeatpolymorphisms. SNPs are not susceptible to artefacts such as stutterbands that can hinder analysis. Stutter bands are frequently encounteredwhen analyzing repeat polymorphisms, and are particularly troublesomewhen analyzing samples such as crime scene samples that may containmixtures of DNA from multiple sources. Another significant advantage ofSNP markers over STR markers is the much shorter length of nucleic acidneeded to score a SNP. For example, STR markers are generally severalhundred base pairs in length. A SNP, on the other hand, comprises asingle nucleotide, and generally a short conserved region on either sideof the SNP position for primer and/or probe binding. This makes SNPsmore amenable to typing in highly degraded or aged biological samplesthat are frequently encountered in forensic casework in which DNA may befragmented into short pieces.

SNPs also are not subject to microvariant and “off-ladder” allelesfrequently encountered when analyzing STR loci. Microvariants aredeletions or insertions within a repeat unit that change the size of theamplified DNA product so that the amplified product does not migrate atthe same rate as reference alleles with normal sized repeat units. Whenseparated by size, such as by electrophoresis on a polyacrylamide gel,microvariants do not align with a reference allelic ladder of standardsized repeat units, but rather migrate between the reference alleles.The reference allelic ladder is used for precise sizing of alleles forallele classification; therefore alleles that do not align with thereference allelic ladder lead to substantial analysis problems.Furthermore, when analyzing multi-allelic repeat polymorphisms,occasionally an allele is found that consists of more or less repeatunits than has been previously seen in the population, or more or lessrepeat alleles than are included in a reference allelic ladder. Thesealleles will migrate outside the size range of known alleles in areference allelic ladder, and therefore are referred to as “off-ladder”alleles. In extreme cases, the allele may contain so few or so manyrepeats that it migrates well out of the range of the reference allelicladder. In this situation, the allele may not even be observed, or, withmultiplex analysis, it may migrate within or close to the size range foranother locus, further confounding analysis.

SNP analysis avoids the problems of microvariants and off-ladder allelesencountered in STR analysis. Importantly, microvariants and off-ladderalleles may provide significant problems, and may be completely missed,when using analysis methods such as oligonucleotide hybridizationarrays, which utilize oligonucleotide probes specific for certain knownalleles. Furthermore, off-ladder alleles and microvariants encounteredwith STR analysis, even when correctly typed, may lead to improperstatistical analysis, since their frequencies in the population aregenerally unknown or poorly characterized, and therefore the statisticalsignificance of a matching genotype may be questionable. All theseadvantages of SNP analysis are considerable in light of the consequencesof most DNA identification cases, which may lead to life imprisonmentfor an individual, or re-association of remains to the family of adeceased individual.

DNA can be isolated from biological samples such as blood, bone, hair,saliva, or semen, and compared with the DNA from a reference source atparticular SNP positions.

Multiple SNP markers can be assayed simultaneously in order to increasethe power of discrimination and the statistical significance of amatching genotype. For example, oligonucleotide arrays can be used togenotype a large number of SNPs simultaneously. The SNPs provided by thepresent invention can be assayed in combination with other polymorphicgenetic markers, such as other SNPs known in the art or STRs, in orderto identify an individual or to associate an individual With aparticular biological sample.

Furthermore, the SNPs provided by the present invention can be genotypedfor inclusion in a database of DNA genotypes, for example, a criminalDNA databank such as the FBI's Combined DNA Index System (CODIS)database. A genotype obtained from a biological sample of unknown sourcecan then be queried against the database to find a matching genotype,with the SNPs of the present invention providing nucleotide positions atwhich to compare the known and unknown DNA sequences for identity.Accordingly, the present invention provides a database comprising novelSNPs or SNP alleles of the present invention (e.g., the database cancomprise information indicating which alleles are possessed byindividual members of a population at one or more novel SNP sites of thepresent invention), such as for use in forensics, biometrics, or otherhuman identification applications. Such a database typically comprises acomputer-based system in which the SNPs or SNP alleles of the presentinvention are recorded on a computer readable medium (see the section ofthe present specification entitled “Computer-Related Embodiments”).

The SNPs of the present invention can also be assayed for use inpaternity testing. The object of paternity testing is usually todetermine whether a male is the father of a child. In most cases, themother of the child is known and thus, the mother's contribution to thechild's genotype can be traced. Paternity testing investigates whetherthe part of the child's genotype not attributable to the mother isconsistent with that of the putative father. Paternity testing can beperformed by analyzing sets of polymorphisms in the putative father andthe child, with the SNPs of the present invention providing nucleotidepositions at which to compare the putative father's and child's DNAsequences for identity. If the set of polymorphisms in the childattributable to the father does not match the set of polymorphisms ofthe putative father, it can be concluded, barring experimental error,that the putative father is not the father of the child. If the set ofpolymorphisms in the child attributable to the father match the set ofpolymorphisms of the putative father, a statistical calculation can beperformed to determine the probability of coincidental match, and aconclusion drawn as to the likelihood that the putative father is thetrue biological father of the child.

In addition to paternity testing, SNPs are also useful for other typesof kinship testing, such as for verifying familial relationships forimmigration purposes, or for cases in which an individual alleges to berelated to a deceased individual in order to claim an inheritance fromthe deceased individual, etc. For further information regarding theutility of SNPs for paternity testing and other types of kinshiptesting, including methods for statistical analysis, see Krawczak,“Informativity assessment for biallelic single nucleotidepolymorphisms”, Electrophoresis 1999 June; 20(8):1676-81.

The use of the SNPs of the present invention for human identificationfurther extends to various authentication systems, commonly referred toas biometric systems, which typically convert physical characteristicsof humans (or other organisms) into digital data. Biometric systemsinclude various technological devices that measure such uniqueanatomical or physiological characteristics as finger, thumb, or palmprints; hand geometry; vein patterning on the back of the hand; bloodvessel patterning of the retina and color and texture of the iris;facial characteristics; voice patterns; signature and typing dynamics;and DNA. Such physiological measurements can be used to verify identityand, for example, restrict or allow access based on the identification.Examples of applications for biometrics include physical area security,computer and network security, aircraft passenger check-in and boarding,financial transactions, medical records access, government benefitdistribution, voting, law enforcement, passports, visas and immigration,prisons, various military applications, and for restricting access toexpensive or dangerous items, such as automobiles or guns (see, forexample, O'Connor, Stanford Technology Law Review and U.S. Pat. No.6,119,096).

Groups of SNPs, particularly the SNPs provided by the present invention,can be typed to uniquely identify an individual for biometricapplications such as those described above. Such SNP typing can readilybe accomplished using, for example, DNA chips/arrays. Preferably, aminimally invasive means for obtaining a DNA sample is utilized. Forexample, PCR amplification enables sufficient quantities of DNA foranalysis to be obtained from buccal swabs or fingerprints, which containDNA-containing skin cells and oils that are naturally transferred duringcontact.

Further information regarding techniques for using SNPs inforensic/human identification applications can be found in, for example,Current Protocols in Human Genetics, John Wiley & Sons, N.Y. (2002),14.1-14.7.

Variant Proteins, Antibodies, Vectors & Host Cells, & Uses Thereof

Variant Proteins Encoded by SNP-Containing Nucleic Acid Molecules

The present invention provides SNP-containing nucleic acid molecules,many of which encode proteins having variant amino acid sequences ascompared to the art-known (i.e., wild-type) proteins. Amino acidsequences encoded by the polymorphic nucleic acid molecules of thepresent invention are provided as SEQ ID NOS:285-568 in Table 1 and theSequence Listing. These variants will generally be referred to herein asvariant proteins/peptides/polypeptides, or polymorphicproteins/peptides/polypeptides of the present invention. The terms“protein”, “peptide”, and “polypeptide” are used herein interchangeably.

A variant protein of the present invention may be encoded by, forexample, a nonsynonymous nucleotide substitution at any one of the cSNPpositions disclosed herein. In addition, variant proteins may alsoinclude proteins whose expression, structure, and/or function is alteredby a SNP disclosed herein, such as a SNP that creates or destroys a stopcodon, a SNP that affects splicing, and a SNP in control/regulatoryelements, e.g. promoters, enhancers, or transcription factor bindingdomains.

As used herein, a protein or peptide is said to be “isolated” or“purified” when it is substantially free of cellular material orchemical precursors or other chemicals. The variant proteins of thepresent invention can be purified to homogeneity or other lower degreesof purity. The level of purification will be based on the intended use.The key feature is that the preparation allows for the desired functionof the variant protein, even if in the presence of considerable amountsof other components.

As used herein, “substantially free of cellular material” includespreparations of the variant protein having less than about 30% (by dryweight) other proteins (i.e., contaminating protein), less than about20% other proteins, less than about 10% other proteins, or less thanabout 5% other proteins. When the variant protein is recombinantlyproduced, it can also be substantially free of culture medium, i.e.,culture medium represents less than about 20% of the volume of theprotein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the variant protein in which it isseparated from chemical precursors or other chemicals that are involvedin its synthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thevariant protein having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

An isolated variant protein may be purified from cells that naturallyexpress it, purified from cells that have been altered to express it(recombinant host cells), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule containing SNP(s) encodingthe variant protein can be cloned into an expression vector, theexpression vector introduced into a host cell, and the variant proteinexpressed in the host cell. The variant protein can then be isolatedfrom the cells by any appropriate purification scheme using standardprotein purification techniques. Examples of these techniques aredescribed in detail below (Sambrook and Russell, 2000, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

The present invention provides isolated variant proteins that comprise,consist of or consist essentially of amino acid sequences that containone or more variant amino acids encoded by one or more codons whichcontain a SNP of the present invention.

Accordingly, the present invention provides variant proteins thatconsist of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists of an amino acid sequencewhen the amino acid sequence is the entire amino acid sequence of theprotein.

The present invention further provides variant proteins that consistessentially of amino acid sequences that contain one or more amino acidpolymorphisms (or truncations or extensions due to creation ordestruction of a stop codon, respectively) encoded by the SNPs providedin Table 1 and/or Table 2. A protein consists essentially of an aminoacid sequence when such an amino acid sequence is present with only afew additional amino acid residues in the final protein.

The present invention further provides variant proteins that compriseamino acid sequences that contain one or more amino acid polymorphisms(or truncations or extensions due to creation or destruction of a stopcodon, respectively) encoded by the SNPs provided in Table 1 and/orTable 2. A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein may contain only the variantamino acid sequence or have additional amino acid residues, such as acontiguous encoded sequence that is naturally associated with it orheterologous amino acid residues. Such a protein can have a fewadditional amino acid residues or can comprise many more additionalamino acids. A brief description of how various types of these proteinscan be made and isolated is provided below.

The variant proteins of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a variant protein operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the variant protein. “Operatively linked”indicates that the coding sequences for the variant protein and theheterologous protein are ligated in-frame. The heterologous protein canbe fused to the N-terminus or C-terminus of the variant protein. Inanother embodiment, the fusion protein is encoded by a fusionpolynucleotide that is synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed and re-amplified to generate a chimeric genesequence (see Ausubel et al., Current Protocols in Molecular Biology,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST protein). A variantprotein-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the variantprotein.

In many uses, the fusion protein does not affect the activity of thevariant protein. The fusion protein can include, but is not limited to,enzymatic fusion proteins, for example, beta-galactosidase fusions,yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-taggedand Ig fusions. Such fusion proteins, particularly poly-His fusions, canfacilitate their purification following recombinant expression. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence. Fusion proteins are further described in, for example, Terpe,“Overview of tag protein fusions: from molecular and biochemicalfundamentals to commercial systems”, Appl Microbiol Biotechnol. 2003January; 60(5):523-33. Epub 2002 Nov. 7; Graddis et al., “Designingproteins that work using recombinant technologies”, Curr PharmBiotechnol. 2002 December; 3(4):285-97; and Nilsson et al., “Affinityfusion strategies for detection, purification, and immobilization ofrecombinant proteins”, Protein Expr Purif 1997 October; 11(1):1-16.

The present invention also relates to further obvious variants of thevariant polypeptides of the present invention, such asnaturally-occurring mature forms (e.g., alleleic variants),non-naturally occurring recombinantly-derived variants, and orthologsand paralogs of such proteins that share sequence homology. Suchvariants can readily be generated using art-known techniques in thefields of recombinant nucleic acid technology and protein biochemistry.It is understood, however, that variants exclude those known in theprior art before the present invention.

Further variants of the variant polypeptides disclosed in Table 1 cancomprise an amino acid sequence that shares at least 70-80%, 80-85%,85-90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identitywith an amino acid sequence disclosed in Table 1 (or a fragment thereof)and that includes a novel amino acid residue (allele) disclosed in Table1 (which is encoded by a novel SNP allele). Thus, the present inventionspecifically contemplates polypeptides that have a certain degree ofsequence variation compared with the polypeptide sequences shown inTable 1, but that contain a novel amino acid residue (allele) encoded bya novel SNP allele disclosed herein. In other words, as long as apolypeptide contains a novel amino acid residue disclosed herein, otherportions of the polypeptide that flank the novel amino acid residue canvary to some degree from the polypeptide sequences shown in Table 1.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the amino acid sequences disclosed hereincan readily be identified as having complete sequence identity to one ofthe variant proteins of the present invention as well as being encodedby the same genetic locus as the variant proteins provided herein.

Orthologs of a variant peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof a variant peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from non-human mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs can be encoded by a nucleic acid sequencethat hybridizes to a variant peptide-encoding nucleic acid moleculeunder moderate to stringent conditions depending on the degree ofrelatedness of the two organisms yielding the homologous proteins.

Variant proteins include, but are not limited to, proteins containingdeletions, additions and substitutions in the amino acid sequence causedby the SNPs of the present invention. One class of substitutions isconserved amino acid substitutions in which a given amino acid in apolypeptide is substituted for another amino acid of likecharacteristics. Typical conservative substitutions are replacements,one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in, forexample, Bowie et al., Science 247:1306-1310 (1990).

Variant proteins can be fully functional or can lack function in one ormore activities, e.g. ability to bind another molecule, ability tocatalyze a substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, truncations or extensions, or a substitution,insertion, inversion, or deletion of a critical residue or in a criticalregion.

Amino acids that are essential for function of a protein can beidentified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science244:1081-1085 (1989)), particularly using the amino acid sequence andpolymorphism information provided in Table 1. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as enzyme activity or in assays such as an in vitro proliferativeactivity. Sites that are critical for binding partner/substrate bindingcan also be determined by structural analysis such as crystallization,nuclear magnetic resonance or photoaffinity labeling (Smith et al., J.Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312(1992)).

Polypeptides can contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Accordingly, the variant proteins of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature polypeptide is fused with anothercompound, such as a compound to increase the half-life of thepolypeptide (e.g., polyethylene glycol), or in which additional aminoacids are fused to the mature polypeptide, such as a leader or secretorysequence or a sequence for purification of the mature polypeptide or apro-protein sequence.

Known protein modifications include, but are not limited to,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such protein modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993); Wold,F., Posttranslational Covalent Modification of Proteins, B. C. Johnson,Ed., Academic Press, New York 1-12 (1983); Seifter et al., Meth.Enzymol. 182: 626-646 (1990); and Rattan et al., Ann. N.Y. Acad. Sci.663:48-62 (1992).

The present invention further provides fragments of the variant proteinsin which the fragments contain one or more amino acid sequencevariations (e.g., substitutions, or truncations or extensions due tocreation or destruction of a stop codon) encoded by one or more SNPsdisclosed herein. The fragments to which the invention pertains,however, are not to be construed as encompassing fragments that havebeen disclosed in the prior art before the present invention.

As used herein, a fragment may comprise at least about 4, 8, 10, 12, 14,16, 18, 20, 25, 30, 50, 100 (or any other number in-between) or morecontiguous amino acid residues from a variant protein, wherein at leastone amino acid residue is affected by a SNP of the present invention,e.g., a variant amino acid residue encoded by a nonsynonymous nucleotidesubstitution at a cSNP position provided by the present invention. Thevariant amino acid encoded by a cSNP may occupy any residue positionalong the sequence of the fragment. Such fragments can be chosen basedon the ability to retain one or more of the biological activities of thevariant protein or the ability to perform a function, e.g., act as animmunogen. Particularly important fragments are biologically activefragments. Such fragments will typically comprise a domain or motif of avariant protein of the present invention, e.g., active site,transmembrane domain, or ligand/substrate binding domain. Otherfragments include, but are not limited to, domain or motif-containingfragments, soluble peptide fragments, and fragments containingimmunogenic structures. Predicted domains and functional sites arereadily identifiable by computer programs well known to those of skillin the art (e.g., PROSITE analysis) (Current Protocols in ProteinScience, John Wiley & Sons, N.Y. (2002)).

Uses of Variant Proteins

The variant proteins of the present invention can be used in a varietyof ways, including but not limited to, in assays to determine thebiological activity of a variant protein, such as in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother type of immune response; as a, reagent (including the labeledreagent) in assays designed to quantitatively determine levels of thevariant protein (or its binding partner) in biological fluids; as amarker for cells or tissues in which it is preferentially expressed(either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); as a target forscreening for a therapeutic agent; and as a direct therapeutic agent tobe administered into a human subject. Any of the variant proteinsdisclosed herein may be developed into reagent grade or kit format forcommercialization as research products. Methods for performing the useslisted above are well known to those skilled in the art (see, e.g.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Sambrook and Russell, 2000, and Methods in Enzymology: Guide toMolecular Cloning Techniques, Academic Press, Berger, S. L. and A. R.Kimmel eds., 1987).

In a specific embodiment of the invention, the methods of the presentinvention include detection of one or more variant proteins disclosedherein. Variant proteins are disclosed in Table 1 and in the SequenceListing as SEQ ID NOS: 285-568. Detection of such proteins can beaccomplished using, for example, antibodies, small molecule compounds,aptamers, ligands/substrates, other proteins or protein fragments, orother protein-binding agents. Preferably, protein detection agents arespecific for a variant protein of the present invention and cantherefore discriminate between a variant protein of the presentinvention and the wild-type protein or another variant form. This cangenerally be accomplished by, for example, selecting or designingdetection agents that bind to the region of a protein that differsbetween the variant and wild-type protein, such as a region of a proteinthat contains one or more amino acid substitutions that is/are encodedby a non-synonymous cSNP of the present invention, or a region of aprotein that follows a nonsense mutation-type SNP that creates a stopcodon thereby leading to a shorter polypeptide, or a region of a proteinthat follows a read-through mutation-type SNP that destroys a stop codonthereby leading to a longer polypeptide in which a portion of thepolypeptide is present in one version of the polypeptide but not theother.

In another specific aspect of the invention, the variant proteins of thepresent invention are used as targets for diagnosing stenosis or fordetermining predisposition to stenosis in a human. Accordingly, theinvention provides methods for detecting the presence of, or levels of,one or more variant proteins of the present invention in a cell, tissue,or organism. Such methods typically involve contacting a test samplewith an agent (e.g., an antibody, small molecule compound, or peptide)capable of interacting with the variant protein such that specificbinding of the agent to the variant protein can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an array, for example, an antibody or aptamer array(arrays for protein detection may also be referred to as “proteinchips”). The variant protein of interest can be isolated from a testsample and assayed for the presence of a variant amino acid sequenceencoded by one or more SNPs disclosed by the present invention. The SNPsmay cause changes to the protein and the corresponding proteinfunction/activity, such as through non-synonymous substitutions inprotein coding regions that can lead to amino acid substitutions,deletions, insertions, and/or rearrangements; formation or destructionof stop codons; or alteration of control elements such as promoters.SNPs may also cause inappropriate post-translational modifications.

One preferred agent for detecting a variant protein in a sample is anantibody capable of selectively binding to a variant form of the protein(antibodies are described in greater detail in the next section). Suchsamples include, for example, tissues, cells, and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject.

In vitro methods for detection of the variant proteins associated withstenosis that are disclosed herein and fragments thereof include, butare not limited to, enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), Western blots, immunoprecipitations,immunofluorescence, and protein arrays/chips (e.g., arrays of antibodiesor aptamers). For further information regarding immunoassays and relatedprotein detection methods, see Current Protocols in Immunology, JohnWiley & Sons, N.Y., and Hage, “Immunoassays”, Anal Chem. 1999 Jun.15;71(12):294R-304R.

Additional analytic methods of detecting amino acid variants include,but are not limited to, altered electrophoretic mobility, alteredtryptic peptide digest, altered protein activity in cell-based orcell-free assay, alteration in ligand or antibody-binding pattern,altered isoelectric point, and direct amino acid sequencing.

Alternatively, variant proteins can be detected in vivo in a subject byintroducing into the subject a labeled antibody (or other type ofdetection reagent) specific for a variant protein. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Other uses of the variant peptides of the present invention are based onthe class or action of the protein. For example, proteins isolated fromhumans and their mammalian orthologs serve as targets for identifyingagents (e.g., small molecule drugs or antibodies) for use in therapeuticapplications, particularly for modulating a biological or pathologicalresponse in a cell or tissue that expresses the protein. Pharmaceuticalagents can be developed that modulate protein activity.

As an alternative to modulating gene expression, therapeutic compoundscan be developed that modulate protein function. For example, many SNPsdisclosed herein affect the amino acid sequence of the encoded protein(e.g., non-synonymous cSNPs and nonsense mutation-type SNPs). Suchalterations in the encoded amino acid sequence may affect proteinfunction, particularly if such amino acid sequence variations occur infunctional protein domains, such as catalytic domains, ATP-bindingdomains, or ligand/substrate binding domains. It is well established inthe art that variant proteins having amino acid sequence variations infunctional domains can cause or influence pathological conditions. Insuch instances, compounds (e.g., small molecule drugs or antibodies) canbe developed that target the variant protein and modulate (e.g., up- ordown-regulate) protein function/activity.

The therapeutic methods of the present invention further include methodsthat target one or more variant proteins of the present invention.Variant proteins can be targeted using, for example, small moleculecompounds, antibodies, aptamers, ligands/substrates, other proteins, orother protein-binding agents. Additionally, the skilled artisan willrecognize that the novel protein variants (and polymorphic nucleic acidmolecules) disclosed in Table 1 may themselves be directly used astherapeutic agents by acting as competitive inhibitors of correspondingart-known proteins (or nucleic acid molecules such as mRNA molecules).

The variant proteins of the present invention are particularly useful indrug screening assays, in cell-based or cell-free systems. Cell-basedsystems can utilize cells that naturally express the protein, a biopsyspecimen, or cell cultures. In one embodiment, cell-based assays involverecombinant host cells expressing the variant protein. Cell-free assayscan be used to detect the ability of a compound to directly bind to avariant protein or to the corresponding SNP-containing nucleic acidfragment that encodes the variant protein.

A variant protein of the present invention, as well as appropriatefragments thereof, can be used in high-throughput screening assays totest candidate compounds for the ability to bind and/or modulate theactivity of the variant protein. These candidate compounds can befurther screened against a protein having normal function (e.g., awild-type/non-variant protein) to further determine the effect of thecompound on the protein activity. Furthermore, these compounds can betested in animal or invertebrate systems to determine in vivoactivity/effectiveness. Compounds can be identified that activate(agonists) or inactivate (antagonists) the variant protein, anddifferent compounds can be identified that cause various degrees ofactivation or inactivation of the variant protein.

Further, the variant proteins can be used to screen a compound for theability to stimulate or inhibit interaction between the variant proteinand a target molecule that normally interacts with the protein. Thetarget can be a ligand, a substrate or a binding partner that theprotein normally interacts with (for example, epinephrine ornorepinephrine). Such assays typically include the steps of combiningthe variant protein with a candidate compound under conditions thatallow the variant protein, or fragment thereof, to interact with thetarget molecule, and to detect the formation of a complex between theprotein and the target or to detect the biochemical consequence of theinteraction with the variant protein and the target, such as any of theassociated effects of signal transduction.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the variant protein thatcompetes for ligand binding. Other candidate compounds include mutantproteins or appropriate fragments containing mutations that affectvariant protein function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) variant protein activity.The assays typically involve an assay of events in the signaltransduction pathway that indicate protein activity. Thus, theexpression of genes that are up or down-regulated in response to thevariant protein dependent signal cascade can be assayed. In oneembodiment, the regulatory region of such genes can be operably linkedto a marker that is easily detectable, such as luciferase.Alternatively, phosphorylation of the variant protein, or a variantprotein target, could also be measured. Any of the biological orbiochemical functions mediated by the variant protein can be used as anendpoint assay. These include all of the biochemical or biologicalevents described herein, in the references cited herein, incorporated byreference for these endpoint assay targets, and other functions known tothose of ordinary skill in the art.

Binding and/or activating compounds can also be screened by usingchimeric variant proteins in which an amino terminal extracellulardomain or parts thereof, an entire transmembrane domain or subregions,and/or the carboxyl terminal intracellular domain or parts thereof, canbe replaced by heterologous domains or subregions. For example, asubstrate-binding region can be used that interacts with a differentsubstrate than that which is normally recognized by a variant protein.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which thevariant protein is derived.

The variant proteins are also useful in competition binding assays inmethods designed to discover compounds that interact with the variantprotein. Thus, a compound can be exposed to a variant protein underconditions that allow the compound to bind or to otherwise interact withthe variant protein. A binding partner, such as ligand, that normallyinteracts with the variant protein is also added to the mixture. If thetest compound interacts with the variant protein or its binding partner,it decreases the amount of complex formed or activity from the variantprotein. This type of assay is particularly useful in screening forcompounds that interact with specific regions of the variant protein(Hodgson, Bio/technology, 1992, Sept 10(9), 973-80).

To perform cell-free drug screening assays, it is sometimes desirable toimmobilize either the variant protein or a fragment thereof, or itstarget molecule, to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay. Any method for immobilizing proteins onmatrices can be used in drug screening assays. In one embodiment, afusion protein containing an added domain allows the protein to be boundto a matrix. For example, glutathione-S-transferase/¹²⁵I fusion proteinscan be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.Louis, Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and a candidatecompound, such as a drug candidate, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads can bewashed to remove any unbound label, and the matrix immobilized andradiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofbound material found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Either the variant protein or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin. Alternatively,antibodies reactive with the variant protein but which do not interferewith binding of the variant protein to its target molecule can bederivatized to the wells of the plate, and the variant protein trappedin the wells by antibody conjugation. Preparations of the targetmolecule and a candidate compound are incubated in the variantprotein-presenting wells and the amount of complex trapped in the wellcan be quantitated. Methods for detecting such complexes, in addition tothose described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with the proteintarget molecule, or which are reactive with variant protein and competewith the target molecule, and enzyme-linked assays that rely ondetecting an enzymatic activity associated with the target molecule.

Modulators of variant protein activity identified according to thesedrug screening assays can be used to treat a subject with a disordermediated by the protein pathway, such as stenosis. These methods oftreatment typically include the steps of administering the modulators ofprotein activity in a pharmaceutical composition to a subject in need ofsuch treatment.

The variant proteins, or fragments thereof, disclosed herein canthemselves be directly used to treat a disorder characterized by anabsence of, inappropriate, or unwanted expression or activity of thevariant protein. Accordingly, methods for treatment include the use of avariant protein disclosed herein or fragments thereof. In yet anotheraspect of the invention, variant proteins can be used as “bait proteins”in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and BrentWO94/10300) to identify other proteins that bind to or interact with thevariant protein and are involved in variant protein activity. Suchvariant protein-binding proteins are also likely to be involved in thepropagation of signals by the variant proteins or variant proteintargets as, for example, elements of a protein-mediated signalingpathway. Alternatively, such variant protein-binding proteins areinhibitors of the variant protein.

The two-hybrid system is based on the modular nature of mosttranscription factors, which typically consist of separable DNA-bindingand activation domains.

Briefly, the assay typically utilizes two different DNA constructs. Inone construct, the gene that codes for a variant protein is fused to agene encoding the DNA binding domain of a known transcription factor(e.g., GAL-4). In the other construct, a DNA sequence, from a library ofDNA sequences, that encodes an unidentified protein (“prey” or “sample”)is fused to a gene that codes for the activation domain of the knowntranscription factor. If the “bait” and the “prey” proteins are able tointeract, in vivo, forming a variant protein-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) that is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected, and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene that encodes the protein that interacts with the variantprotein.

Antibodies Directed to Variant Proteins

The present invention also provides antibodies that selectively bind tothe variant proteins disclosed herein and fragments thereof. Suchantibodies may be used to quantitatively or qualitatively detect thevariant proteins of the present invention. As used herein, an antibodyselectively binds a target variant protein when it binds the variantprotein and does not significantly bind to non-variant proteins, i.e.,the antibody does not significantly bind to normal, wild-type, orart-known proteins that do not contain a variant amino acid sequence dueto one or more SNPs of the present invention (variant amino acidsequences may be due to, for example, nonsynonymous cSNPs, nonsense SNPsthat create a stop codon, thereby causing a truncation of a polypeptideor SNPs that cause read-through mutations resulting in an extension of apolypeptide).

As used herein, an antibody is defined in terms consistent with thatrecognized in the art: they are multi-subunit proteins produced by anorganism in response to an antigen challenge. The antibodies of thepresent invention include both monoclonal antibodies and polyclonalantibodies, as well as antigen-reactive proteolytic fragments of suchantibodies, such as Fab, F(ab)′₂, and Fv fragments. In addition, anantibody of the present invention further includes any of a variety ofengineered antigen-binding molecules such as a chimeric antibody (U.S.Pat. Nos. 4,816,567 and 4,816,397; Morrison et al., Proc. Natl. Acad.Sci. USA, 81:6851, 1984; Neuberger et al., Nature 312:604, 1984), ahumanized antibody (U.S. Pat. Nos. 5,693,762; 5,585,089; and 5,565,332),a single-chain Fv (U.S. Pat. No. 4,946,778; Ward et al., Nature 334:544,1989), a bispecific antibody with two binding specificities (Segal etal., J. Immunol. Methods 248:1, 2001; Carter, J. Immunol. Methods 248:7,2001), a diabody, a triabody, and a tetrabody (Todorovska et al., J.Immunol. Methods, 248:47, 2001), as well as a Fab conjugate (dimer ortrimer), and a minibody.

Many methods are known in the art for generating and/or identifyingantibodies to a given target antigen (Harlow, Antibodies, Cold SpringHarbor Press, (1989)). In general, an isolated peptide (e.g., a variantprotein of the present invention) is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit, hamster ormouse. Either a full-length protein, an antigenic peptide fragment(e.g., a peptide fragment containing a region that varies between avariant protein and a corresponding wild-type protein), or a fusionprotein can be used. A protein used as an immunogen may benaturally-occurring, synthetic or recombinantly produced, and may beadministered in combination with an adjuvant, including but not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substance such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and the like.

Monoclonal antibodies can be produced by hybridoma technology (Kohlerand Milstein, Nature, 256:495, 1975), which immortalizes cells secretinga specific monoclonal antibody. The immortalized cell lines can becreated in vitro by fusing two different cell types, typicallylymphocytes, and tumor cells. The hybridoma cells may be cultivated invitro or in vivo. Additionally, fully human antibodies can be generatedby transgenic animals (He et al., J. Immunol., 169:595, 2002). Fd phageand Fd phagemid technologies may be used to generate and selectrecombinant antibodies in vitro (Hoogenboom and Chames, Immunol. Today21:371, 2000; Liu et al., J. Mol. Biol. 315:1063, 2002). Thecomplementarity-determining regions of an antibody can be identified,and synthetic peptides corresponding to such regions may be used tomediate antigen binding (U.S. Pat. No. 5,637,677).

Antibodies are preferably prepared against regions or discrete fragmentsof a variant protein containing a variant amino acid sequence ascompared to the corresponding wild-type protein (e.g., a region of avariant protein that includes an amino acid encoded by a nonsynonymouscSNP, a region affected by truncation caused by a nonsense SNP thatcreates a stop codon, or a region resulting from the destruction of astop codon due to read-through mutation caused by a SNP). Furthermore,preferred regions will include those involved in function/activityand/or protein/binding partner interaction. Such fragments can beselected on a physical property, such as fragments corresponding toregions that are located on the surface of the protein, e.g.,hydrophilic regions, or can be selected based on sequence uniqueness, orbased on the position of the variant amino acid residue(s) encoded bythe SNPs provided by the present invention. An antigenic fragment willtypically comprise at least about 8-10 contiguous amino acid residues inwhich at least one of the amino acid residues is an amino acid affectedby a SNP disclosed herein. The antigenic peptide can comprise, however,at least 12, 14, 16, 20, 25, 50, 100 (or any other number in-between) ormore amino acid residues, provided that at least one amino acid isaffected by a SNP disclosed herein.

Detection of an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody or an antigen-reactivefragment thereof to a detectable substance. Detectable substancesinclude, but are not limited to, various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies, particularly the use of antibodies as therapeutic agents,are reviewed in: Morgan, “Antibody therapy for Alzheimer's disease”,Expert Rev Vaccines. 2003 February; 2(1):53-9; Ross et al., “Anticancerantibodies”, Am J Clin Pathol. 2003 April; 119(4):472-85; Goldenberg,“Advancing role of radiolabeled antibodies in the therapy of cancer”,Cancer Immunol Immunother. 2003 May; 52(5):281-96. Epub 2003 Mar 11;Ross et al., “Antibody-based therapeutics in oncology”, Expert RevAnticancer Ther. 2003 February; 3(1):107-21; Cao et al., “Bispecificantibody conjugates in therapeutics”, Adv Drug Deliv Rev. 2003 Feb.10;55(2):171-97; von Mehrenet al., “Monoclonal antibody therapy forcancer”, Annu Rev Med. 2003;54:343-69. Epub 2001 Dec. 3; Hudson et al.,“Engineered antibodies”, Nat Med. 2003 January; 9(1): 129-34; Brekke etal., “Therapeutic antibodies for human diseases at the dawn of thetwenty-first century”, Nat Rev Drug Discov. 2003 January; 2(1):52-62(Erratum in: Nat Rev Drug Discov. 2003 March; 2(3):240); Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems”, Curr Opin Biotechnol. 2002 December; 13(6):625-9; Andreakos etal., “Monoclonal antibodies in 100, immune and inflammatory diseases”,Curr Opin Biotechnol. 2002 December; 13(6):615-20; Kellermann et al.,“Antibody discovery: the use of transgenic mice to generate humanmonoclonal antibodies for therapeutics”, Curr Opin Biotechnol. 2002December; 13(6):593-7; Pini et al., “Phage display and colony filterscreening for high-throughput selection of antibody libraries”, CombChem High Throughput Screen. 2002 November; 5(7):503-10; Batra et al.,“Pharmacokinetics and biodistribution of genetically engineeredantibodies”, Curr Opin Biotechnol. 2002 December; 13(6):603-8; andTangri et al., “Rationally engineered proteins or antibodies with absentor reduced immunogenicity”, Curr Med Chem. 2002 December; 9(24):2191-9.

Uses of Antibodies

Antibodies can be used to isolate the variant proteins of the presentinvention from a natural cell source or from recombinant host cells bystandard techniques, such as affinity chromatography orimmunoprecipitation. In addition, antibodies are useful for detectingthe presence of a variant protein of the present invention in cells ortissues to determine the pattern of expression of the variant proteinamong various tissues in an organism and over the course of normaldevelopment or disease progression. Further, antibodies can be used todetect variant protein in situ, in vitro, in a bodily fluid, or in acell lysate or supernatant in order to evaluate the amount and patternof expression. Also, antibodies can be used to assess abnormal tissuedistribution, abnormal expression during development, or expression inan abnormal condition, such as stenosis. Additionally, antibodydetection of circulating fragments of the full-length variant proteincan be used to identify turnover.

Antibodies to the variant proteins of the present invention are alsouseful in pharmacogenomic analysis. Thus, antibodies against variantproteins encoded by alternative SNP alleles can be used to identifyindividuals that require modified treatment modalities.

Further, antibodies can be used to assess expression of the variantprotein in disease states such as in active stages of the disease or inan individual with a predisposition to a disease related to theprotein's function, particularly stenosis. Antibodies specific for avariant protein encoded by a SNP-containing nucleic acid molecule of thepresent invention can be used to assay for the presence of the variantprotein, such as to screen for predisposition to stenosis as indicatedby the presence of the variant protein.

Antibodies are also useful as diagnostic tools for evaluating thevariant proteins in conjunction with analysis by electrophoreticmobility, isoelectric point, tryptic peptide digest, and other physicalassays well known in the art.

Antibodies are also useful for tissue typing. Thus, where a specificvariant protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

Antibodies can also be used to assess aberrant subcellular localizationof a variant protein in cells in various tissues. The diagnostic usescan be applied, not only in genetic testing, but also in monitoring atreatment modality. Accordingly, where treatment is ultimately aimed atcorrecting the expression level or the presence of variant protein oraberrant tissue distribution or developmental expression of a variantprotein, antibodies directed against the variant protein or relevantfragments can be used to monitor therapeutic efficacy.

The antibodies are also useful for inhibiting variant protein function,for example, by blocking the binding of a variant protein to a bindingpartner. These uses can also be applied in a therapeutic context inwhich treatment involves inhibiting a variant protein's function. Anantibody can be used, for example, to block or competitively inhibitbinding, thus modulating (agonizing or antagonizing) the activity of avariant protein. Antibodies can be prepared against specific variantprotein fragments containing sites required for function or against anintact variant protein that is associated with a cell or cell membrane.For in vivo administration, an antibody may be linked with an additionaltherapeutic payload such as a radionuclide, an enzyme, an immunogenicepitope, or a cytotoxic agent. Suitable cytotoxic agents include, butare not limited to, bacterial toxin such as diphtheria, and plant toxinsuch as ricin. The in vivo half-life of an antibody or a fragmentthereof may be lengthened by pegylation through conjugation topolyethylene glycol (Leong et al., Cytokine 16:106, 2001).

The invention also encompasses kits for using antibodies, such as kitsfor detecting the presence of a variant protein in a test sample. Anexemplary kit can comprise antibodies such as a labeled or labelableantibody and a compound or agent for detecting variant proteins in abiological sample; means for determining the amount, or presence/absenceof variant protein in the sample; means for comparing the amount ofvariant protein in the sample with a standard; and instructions for use.

Vectors and Host Cells

The present invention also provides vectors containing theSNP-containing nucleic acid molecules described herein. The term“vector” refers to a vehicle, preferably a nucleic acid molecule, whichcan transport a SNP-containing nucleic acid molecule. When the vector isa nucleic acid molecule, the SNP-containing nucleic acid molecule can becovalently linked to the vector nucleic acid. Such vectors include, butare not limited to, a plasmid, single or double stranded phage, a singleor double stranded RNA or DNA viral vector, or artificial chromosome,such as a BAC, PAC, YAC, or MAC.

A vector can be maintained in a host cell as an extrachromosomal elementwhere it replicates and produces additional copies of the SNP-containingnucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the SNP-containingnucleic acid molecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the SNP-containingnucleic acid molecules. The vectors can function in prokaryotic oreukaryotic cells or in both (shuttle vectors).

Expression vectors typically contain cis-acting regulatory regions thatare operably linked in the vector to the SNP-containing nucleic acidmolecules such that transcription of the SNP-containing nucleic acidmolecules is allowed in a host cell. The SNP-containing nucleic acidmolecules can also be introduced into the host cell with a separatenucleic acid molecule capable of affecting transcription. Thus, thesecond nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the SNP-containing nucleic acid molecules from thevector. Alternatively, a trans-acting factor may be supplied by the hostcell. Finally, a trans-acting factor can be produced from the vectoritself. It is understood, however, that in some embodiments,transcription and/or translation of the nucleic acid molecules can occurin a cell-free system.

The regulatory sequences to which the SNP-containing nucleic acidmolecules described herein can be operably linked include promoters fordirecting mRNA transcription. These include, but are not limited to, theleft promoter from bacteriophage λ, the lac, TRP, and TAC promoters fromE. coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region, aribosome-binding site for translation. Other regulatory control elementsfor expression include initiation and termination codons as well aspolyadenylation signals. A person of ordinary skill in the art would beaware of the numerous regulatory sequences that are useful in expressionvectors (see, e.g., Sambrook and Russell, 2000, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

A variety of expression vectors can be used to express a SNP-containingnucleic acid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example, vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors can also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g., cosmids and phagemids. Appropriate cloning andexpression vectors for prokaryotic and eukaryotic hosts are described inSambrook and Russell, 2000, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The regulatory sequence in a vector may provide constitutive expressionin one or more host cells (e.g., tissue specific expression) or mayprovide for inducible expression in one or more cell types such as bytemperature, nutrient additive, or exogenous factor, e.g., a hormone orother ligand. A variety of vectors that provide constitutive orinducible expression of a nucleic acid sequence in prokaryotic andeukaryotic host cells are well known to those of ordinary skill in theart.

A SNP-containing nucleic acid molecule can be inserted into the vectorby methodology well-known in the art. Generally, the SNP-containingnucleic acid molecule that will ultimately be expressed is joined to anexpression vector by cleaving the SNP-containing nucleic acid moleculeand the expression vector with one or more restriction enzymes and thenligating the fragments together. Procedures for restriction enzymedigestion and ligation are well known to those of ordinary skill in theart.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques.

Bacterial host cells include, but are not limited to, E. coli,Streptomyces, and Salmonella typhimurium. Eukaryotic host cells include,but are not limited to, yeast, insect cells such as Drosophila, animalcells such as COS and CHO cells, and plant cells.

As described herein, it may be desirable to express the variant peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the variant peptides. Fusion vectorscan, for example, increase the expression of a recombinant protein,increase the solubility of the recombinant protein, and aid in thepurification of the protein by acting, for example, as a ligand foraffinity purification. A proteolytic cleavage site may be introduced atthe junction of the fusion moiety so that the desired variant peptidecan ultimately be separated from the fusion moiety. Proteolytic enzymessuitable for such use include, but are not limited to, factor Xa,thrombin, and enterokinase. Typical fusion expression vectors includepGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

Recombinant protein expression can be maximized in a bacterial host byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe SNP-containing nucleic acid molecule of interest can be altered toprovide preferential codon usage for a specific host cell, for example,E. coli (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

The SNP-containing nucleic acid molecules can also be expressed byexpression vectors that are operative in yeast. Examples of vectors forexpression in yeast (e.g., S. cerevisiae) include pYepSec1 (Baldari, etal., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al., Cell30:933-943(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), andpYES2 (Invitrogen Corporation, San Diego, Calif.).

The SNP-containing nucleic acid molecules can also be expressed ininsect cells using, for example, baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., Sf9 cells) include the pAc series (Smith et al.,Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al.,Virology 170:31-39 (1989)).

In certain embodiments of the invention, the SNP-containing nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

The invention also encompasses vectors in which the SNP-containingnucleic acid molecules described herein are cloned into the vector inreverse orientation, but operably linked to a regulatory sequence thatpermits transcription of antisense RNA. Thus, an antisense transcriptcan be produced to the SNP-containing nucleic acid sequences describedherein, including both coding and non-coding regions. Expression of thisantisense RNA is subject to each of the parameters described above inrelation to expression of the sense RNA (regulatory sequences,constitutive or inducible expression, tissue-specific expression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include, for example,prokaryotic cells, lower eukaryotic cells such as yeast, othereukaryotic cells such as insect cells, and higher eukaryotic cells suchas mammalian cells.

The recombinant host cells can be prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to persons of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those described in Sambrook and Russell, 2000,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Host cells can contain more than one vector. Thus, differentSNP-containing nucleotide sequences can be introduced in differentvectors into the same cell. Similarly, the SNP-containing nucleic acidmolecules can be introduced either alone or with other nucleic acidmolecules that are not related to the SNP-containing nucleic acidmolecules, such as those providing trans-acting factors for expressionvectors. When more than one vector is introduced into a cell, thevectors can be introduced independently, co-introduced, or joined to thenucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication can occur in host cells that provide functionsthat complement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be inserted in the same vector that containsthe SNP-containing nucleic acid molecules described herein or may be ina separate vector. Markers include, for example, tetracycline orampicillin-resistance genes for prokaryotic host cells, anddihydrofolate reductase or neomycin resistance genes for eukaryotic hostcells. However, any marker that provides selection for a phenotypictrait can be effective.

While the mature variant proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these variant proteins using RNA derivedfrom the DNA constructs described herein.

Where secretion of the variant protein is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such asG-protein-coupled receptors (GPCRs), appropriate secretion signals canbe incorporated into the vector. The signal sequence can be endogenousto the peptides or heterologous to these peptides.

Where the variant protein is not secreted into the medium, the proteincan be isolated from the host cell by standard disruption procedures,including freeze/thaw, sonication, mechanical disruption, use of lysingagents, and the like. The variant protein can then be recovered andpurified by well-known purification methods including, for example,ammonium sulfate precipitation, acid extraction, anion or cationicexchange chromatography, phosphocellulose chromatography,hydrophobic-interaction chromatography, affinity chromatography,hydroxylapatite chromatography, lectin chromatography, or highperformance liquid chromatography.

It is also understood that, depending upon the host cell in whichrecombinant production of the variant proteins described herein occurs,they can have various glycosylation patterns, or may benon-glycosylated, as when produced in bacteria. In addition, the variantproteins may include an initial modified methionine in some cases as aresult of a host-mediated process.

For further information regarding vectors and host cells, see CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y.

Uses of Vectors and Host Cells, and Transgenic Animals

Recombinant host cells that express the variant proteins describedherein have a variety of uses. For example, the cells are useful forproducing a variant protein that can be further purified into apreparation of desired amounts of the variant protein or fragmentsthereof. Thus, host cells containing expression vectors are useful forvariant protein production.

Host cells are also useful for conducting cell-based assays involvingthe variant protein or variant protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a variant protein is useful forassaying compounds that stimulate or inhibit variant protein function.Such an ability of a compound to modulate variant protein function maynot be apparent from assays of the compound on the native/wild-typeprotein, or from cell-free assays of the compound. Recombinant hostcells are also useful for assaying functional alterations in the variantproteins as compared with a known function.

Genetically-engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably anon-human mammal, for example, a rodent, such as a rat or mouse, inwhich one or more of the cells of the animal include a transgene. Atransgene is exogenous DNA containing a SNP of the present inventionwhich is integrated into the genome of a cell from which a transgenicanimal develops and which remains in the genome of the mature animal inone or more of its cell types or tissues. Such animals are useful forstudying the function of a variant protein in vivo, and identifying andevaluating modulators of variant protein activity. Other examples oftransgenic animals include, but are not limited to, non-human primates,sheep, dogs, cows, goats, chickens, and amphibians. Transgenic non-humanmammals such as cows and goats can be used to produce variant proteinswhich can be secreted in the animal's milk and then recovered.

A transgenic animal can be produced by introducing a SNP-containingnucleic acid molecule into the male pronuclei of a fertilized oocyte,e.g., by microinjection or retroviral infection, and allowing the oocyteto develop in a pseudopregnant female foster animal. Any nucleic acidmolecules that contain one or more SNPs of the present invention canpotentially be introduced as a transgene into the genome of a non-humananimal.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the variant protein in particularcells or tissues.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described in, for example, U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al., and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes a non-human animal in which the entire animal or tissuesin the animal have been produced using the homologously recombinant hostcells described herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. PNAS 89:6232-6236 (1992)).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991)). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein are generally needed. Such animalscan be provided through the construction of “double” transgenic animals,e.g., by mating two transgenic animals, one containing a transgeneencoding a selected variant protein and the other containing a transgeneencoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in, for example, Wilmut, I.et al. Nature 385:810-813 (1997) and PCT International Publication Nos.WO 97/07668 and WO 97/07669. In brief, a cell (e.g., a somatic cell)from the transgenic animal can be isolated and induced to exit thegrowth cycle and enter G₀ phase. The quiescent cell can then be fused,e.g., through the use of electrical pulses, to an enucleated oocyte froman animal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell (e.g., a somatic cell) isisolated.

Transgenic animals containing recombinant cells that express the variantproteins described herein are useful for conducting the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could influence ligand orsubstrate binding, variant protein activation, signal transduction, orother processes or interactions, may not be evident from in vitrocell-free or cell-based assays. Thus, non-human transgenic animals ofthe present invention may be used to assay in vivo variant proteinfunction as well as the activities of a therapeutic agent or compoundthat modulates variant protein function/activity or expression. Suchanimals are also suitable for assessing the effects of null mutations(i.e., mutations that substantially or completely eliminate one or morevariant protein functions).

For further information regarding transgenic animals, see Houdebine,“Antibody manufacture in transgenic animals and comparisons with othersystems”, Curr Opin Biotechnol. 2002 December; 13(6):625-9; Petters etal., “Transgenic animals as models for human disease”, Transgenic Res.2000;9(4-5):347-51; discussion 345-6; Wolf et al., “Use of transgenicanimals in understanding molecular mechanisms of toxicity”, J PharmPharmacol. 1998 June; 50(6):567-74; Echelard, “Recombinant proteinproduction in transgenic animals”, Curr Opin Biotechnol. 1996 October;7(5):536-40; Houdebine, “Transgenic animal bioreactors”, Transgenic Res.2000;9(4-5):305-20; Pirity et al., “Embryonic stem cells, creatingtransgenic animals”, Methods Cell Biol. 1998;57:279-93; and Robl et al.,“Artificial chromosome vectors and expression of complex proteins intransgenic animals”, Theriogenology. 2003 Jan. 1;59(1):107-13.

Computer-Related Embodiments

The SNPs provided in the present invention may be “provided” in avariety of mediums to facilitate use thereof. As used in this section,“provided” refers to a manufacture, other than an isolated nucleic acidmolecule, that contains SNP information of the present invention. Such amanufacture provides the SNP information in a form that allows a skilledartisan to examine the manufacture using means not directly applicableto examining the SNPs or a subset thereof as they exist in nature or inpurified form. The SNP information that may be provided in such a formincludes any of the SNP information provided by the present inventionsuch as, for example, polymorphic nucleic acid and/or amino acidsequence information such as SEQ ID NOS:1-284, SEQ ID NOS:285-568, SEQID NOS:5608-5780, SEQ ID NOS:569-5607, and SEQ ID NOS:5781-26,130;information about observed SNP alleles, alternative codons, populations,allele frequencies, SNP types, and/or affected proteins; or any otherinformation provided by the present invention in Tables 1-2 and/or theSequence Listing.

In one application of this embodiment, the SNPs of the present inventioncan be recorded on a computer readable medium. As used herein, “computerreadable medium” refers to any medium that can be read and accesseddirectly by a computer. Such media include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as CD-ROM; electricalstorage media such as RAM and ROM; and hybrids of these categories suchas magnetic/optical storage media. A skilled artisan can readilyappreciate how any of the presently known computer readable media can beused to create a manufacture comprising computer readable medium havingrecorded thereon a nucleotide sequence of the present invention. Onesuch medium is provided with the present application, namely, thepresent application contains computer readable medium (CD-R) that hasnucleic acid sequences (and encoded protein sequences) containing SNPsprovided/recorded thereon in ASCII text format in a Sequence Listingalong with accompanying Tables that contain detailed SNP and sequenceinformation (transcript sequences are provided as SEQ ID NOS:11-284,protein sequences are provided as SEQ ID NOS:285-568, genomic sequencesare provided as SEQ ID NOS:5608-5780, transcript-based context sequencesare provided as SEQ ID NOS:569-5607, and genomic-based context sequencesare provided as SEQ ID NOS:5781-26,130).

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate manufactures comprising the SNP informationof the present invention.

A variety of data storage structures are available to a skilled artisanfor creating a computer readable medium having recorded thereon anucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotide/aminoacid sequence information of the present invention on computer readablemedium. For example, the sequence information can be represented in aword processing text file, formatted in commercially-available softwaresuch as WordPerfect and Microsoft Word, represented in the form of anASCII file, or stored in a database application, such as OB2, Sybase,Oracle, or the like. A skilled artisan can readily adapt any number ofdata processor structuring formats (e.g., text file or database) inorder to obtain computer readable medium having recorded thereon the SNPinformation of the present invention.

By providing the SNPs of the present invention in computer readableform, a skilled artisan can routinely access the SNP information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. Examples of publicly available computersoftware include BLAST (Altschul et at, J. Mol. Biol. 215:403-410(1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) searchalgorithms.

The present invention further provides systems, particularlycomputer-based systems, which contain the SNP information describedherein. Such systems may be designed to store and/or analyze informationon, for example, a large number of SNP positions, or information on SNPgenotypes from a large number of individuals. The SNP information of thepresent invention represents a valuable information source. The SNPinformation of the present invention stored/analyzed in a computer-basedsystem may be used for such computer-intensive applications asdetermining or analyzing SNP allele frequencies in a population, mappingdisease genes, genotype-phenotype association studies, grouping SNPsinto haplotypes, correlating SNP haplotypes with response to particulardrugs, or for various other bioinformatic, pharmacogenomic, drugdevelopment, or human identification/forensic applications.

As used herein, “a computer-based system” refers to the hardware means,software means, and data storage means used to analyze the SNPinformation of the present invention. The minimum hardware means of thecomputer-based systems of the present invention typically comprises acentral processing unit (CPU), input means, output means, and datastorage means. A skilled artisan can readily appreciate that any one ofthe currently available computer-based systems are suitable for use inthe present invention. Such a system can be changed into a system of thepresent invention by utilizing the SNP information provided on the CD-R,or a subset thereof, without any experimentation.

As stated above, the computer-based systems of the present inventioncomprise a data storage means having stored therein SNPs of the presentinvention and the necessary hardware means and software means forsupporting and implementing a search means. As used herein, “datastorage means” refers to memory which can store SNP information of thepresent invention, or a memory access means which can accessmanufactures having recorded thereon the SNP information of the presentinvention.

As used herein, “search means” refers to one or more programs oralgorithms that are implemented on the computer-based system to identifyor analyze SNPs in a target sequence based on the SNP information storedwithin the data storage means. Search means can be used to determinewhich nucleotide is present at a particular SNP position in the targetsequence. As used herein, a “target sequence” can be any DNA sequencecontaining the SNP position(s) to be searched or queried.

As used herein, “a target structural motif,” or “target motif,” refersto any rationally selected sequence or combination of sequencescontaining a SNP position in which the sequence(s) is chosen based on athree-dimensional configuration that is formed upon the folding of thetarget motif. There are a variety of target motifs known in the art.Protein target motifs include, but are not limited to, enzymatic activesites and signal sequences. Nucleic acid target motifs include, but arenot limited to, promoter sequences, hairpin structures, and inducibleexpression elements (protein binding sequences).

A variety of structural formats for the input and output means can beused to input and output the information in the computer-based systemsof the present invention. An exemplary format for an output means is adisplay that depicts the presence or absence of specified nucleotides(alleles) at particular SNP positions of interest. Such presentation canprovide a rapid, binary scoring system for many SNPs simultaneously.

One exemplary embodiment of a computer-based system comprising SNPinformation of the present invention is provided in FIG. 1. FIG. 1provides a block diagram of a computer system 102 that can be used toimplement the present invention. The computer system 102 includes aprocessor 106 connected to a bus 104. Also connected to the bus 104 area main memory 108 (preferably implemented as random access memory, RAM)and a variety of secondary storage devices 110, such as a hard drive 112and a removable medium storage device 114. The removable medium storagedevice 114 may represent, for example, a floppy disk drive, a CD-ROMdrive, a magnetic tape drive, etc. A removable storage medium 116 (suchas a floppy disk, a compact disk, a magnetic tape, etc.) containingcontrol logic and/or data recorded therein may be inserted into theremovable medium storage device 114. The computer system 102 includesappropriate software for reading the control logic and/or the data fromthe removable storage medium 116 once inserted in the removable mediumstorage device 114.

The SNP information of the present invention may be stored in awell-known manner in the main memory 108, any of the secondary storagedevices 110, and/or a removable storage medium 116. Software foraccessing and processing the SNP information (such as SNP scoring tools,search tools, comparing tools, etc.) preferably resides in main memory108 during execution.

EXAMPLES Statistical Analysis of SNP Association with Coronary Stenosis

A case-control genetic study to determine the association of SNPs in thehuman genome with coronary stenosis was carried out using genomic DNAextracted from 2 independently collected case-control sample sets. Asample set from the University of California, San Francisco (UCSF),referred to as “Sample Set 1”, consisted of DNA from 1,654 Caucasianpatients with varying degrees of coronary artery stenosis as evidencedby coronary angiography. A sample set from the Cleveland Clinic (CCF),referred to as “Sample Set 2”, consisted of DNA from 1,501 Caucasianpatients with very little or severe coronary artery stenosis, alsoevidenced by coronary angiography. All samples were obtained from peoplebetween the ages of 18 to 75. All individuals who were included in eachstudy had signed a written informed consent form. The study protocolswere IRB approved.

DNA was extracted from blood samples using conventional DNA extractionmethods such as the QIA-amp kit from Qiagen. SNP markers in theextracted DNA samples were analyzed by genotyping. While some sampleswere individually genotyped, the same samples were also used for poolingstudies, in which DNA samples from about 50 individuals were pooled, andallele frequencies were determined in pooled DNA. Genotypes and poolallele frequencies were obtained using a PRISM 7900HT sequence detectionPCR system (Applied Biosystems, Foster City, Calif.) by allele-specificPCR, similar to the method described by Germer et al (Germer S., HollandM. J., Higuchi R. 2000, Genome Res. 10: 258-266). Primers for theallele-specific PCR reactions are provided in Table 5.

Genotype or allele frequency results from 287 SNPs in Sample Set 1 and177 SNPs in Sample Set 2 were analyzed for association with coronarystenosis. Analysis of the Sample Set 1 data was performed by placingindividuals into groups based on quartiles of the sum score and usingthe two extreme quartiles as a binary endpoint. For Sample Set 2samples, analysis was done in two ways. One method placed individualsinto groups based on quartiles of the sum scores and used the twoextreme quartiles as a binary endpoint. The other method placedindividuals into groups of sum score “0” (the control group) or the sumscore “>0” (the case group). The “sum score” is a measure of the overallextent of coronary artery stenosis and was determined by summingmeasures of percent stenosis from various arterial locations. Thepercent stenosis was determined from images obtained using coronaryangiography. The case and control groups were further stratified by sex(F, M), age (tertile 1, 2, or 3) and smoking status (“never smoked” and“ever smoked”). The allele or genotype frequencies for the tested SNPswere obtained, and compared between cases and controls. No multipletesting corrections were made.

Several tests of association were calculated for both unstratified andstratified settings: 1) Fisher's exact test or asymptotic chi-squaretest for allelic association, 2) asymptotic chi-square test of genotypicassociation of two different modes of inheritance: dominant andrecessive, and 3) Armitage trendtest for the additive mode of genotypicassociation.

Effect sizes were estimated through genotypic or allelic odds ratios,including 95% confidence intervals. The reported allele (Allele1) orgenotype may be under-represented in cases (with a lower frequency incases than in controls, indicating that the reported allele or genotypeis associated with decreased risk and the other allele or genotype is arisk factor for disease) or over-represented in cases (indicating thatthe reported allele or genotype is a risk factor for disease).

The replicated coronary stenosis markers are reported in Table 6. A SNPis considered a replicated marker if the association analyses in twostudies showed that the risk allele is the same, the p-values are eachless than or equal to 0.05, and the significant association is seen ineither the same stratum or in a stratum and its substratum. Table 6 alsoincludes SNPs for which Cochran Mantel Haenszel test showed that theadjusted p-value of the meta analysis was less than 0.05, although thep-value for the individual sample sets might be greater than 0.05. Table7 provides SNPs having a significant association (p-value of less thanor equal to 0.05) with coronary stenosis in either the Sample Set 1 orthe Sample Set 2 study.

An example of a replicated marker, where the homozygous reported alleleis associated with a decreased risk for coronary stenosis is hCV1608777(Table 6). Individuals in the second age tertile (Age T2) with 2 copiesof the reported allele of HCV1608777 (Mode Rec) showed significantassociation (p-values 0.0344 and 0.0076) with decreased risk (oddsratios of 0.51 and 0.19 times of the reference) when compared to thosecarrying one or none of the reported allele (heterozygotes and majorhomozygotes) in both the Sample Set 1 and the Sample Set 2 studies.

An example of a replicated marker, where the reported allele isassociated with increased risk for coronary stenosis is hCV16165996(Table 6). Among the whole study population (Strata=ALL), carriers ofone or two copies of the reported allele (Mode Dom) of HCV16165996showed significant association (p-values of 0.0306 and 0.0037) withcoronary stenosis with an increased risk (odds ratios of 1.31 and 1.69)compared with those carrying none of the reported allele (majorhomozygotes) in both the Sample Set 1 and the Sample Set 2 studies.

All publications and patents cited in this specification are hereinincorporated by reference in their entirety. Various modifications andvariations of the described compositions, methods and systems of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments andcertain working examples, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the above-described modes for carryingout the invention that are obvious to those skilled in the field ofmolecular biology, genetics and related fields are intended to be withinthe scope of the following claims.

TABLE 5 hCV Alleles Sequence A (allele-specific primer) Sequence B(allele-specific primer) Sequence C (common primer) hCV1129436 C/ATTCCAGGGTATATCTCAGAGC (SEQ ID NO: 26131) GTTCCAGGGTATATCTCAGAGA (SEQ IDNO: 26132) TTGAAAGAGTGTGAGCAAGATC (SEQ ID NO: 26133) hCV11592758 T/CCATCCAACAGCTCTTCTATCAT (SEQ ID NO: 26134) CATCCAACAGCTCTTCTATCAC (SEQ IDNO: 26135) CAAACATCCGAGGACAAG (SEQ ID NO: 26136) hCV11623862 T/GCACCTTTCAGGGTTGAAGT (SEQ ID NO: 26137) ACCTTTCAGGGTTGAAGG (SEQ ID NO:26138) GCTCCTCATCTCTTCTCTGTATC (SEQ ID NO: 26139) hCV11660791 T/CTGAGGTCACCTCTAGCAAGTT (SEQ ID NO: 26140) TGAGGTCACCTCTAGCAAGTC (SEQ IDNO: 26141) TCATTGCATGCTTTCATTTATAA (SEQ ID NO: 26142) hCV11972321 G/AGGTTCTGACATGACTGTGACAG (SEQ ID NO: 26143) GTTCTGACATGACTGTGACAA (SEQ IDNO: 26144) CCCCAACCCAAGGTTTAC (SEQ ID NO: 26145) hCV11975277 C/AGAATGGCCACTGGTCAC (SEQ ID NO: 26146) GAATGGCCACTGGTCAA (SEQ ID NO:26147) ACACAGGGAAGAAACACTGAT (SEQ ID NO: 26148) hCV1202883 G/AGCGTGATGATGAAATCGG (SEQ ID NO: 26149) GCGTGATGATGAAATCGA (SEQ ID NO:26150) AGCCTCTCCTGACTGTCATC (SEQ ID NO: 26151) hCV1309246 T/CGCTTCCTGGCAACACTTT (SEQ ID NO: 26152) GCTTCCTGGCAACACTTC (SEQ ID NO:26153) CAGTCACAGCTTGCAATCATA (SEQ ID NO: 26154) hCV1345898 C/TCAGTTTTCCATGGGTTCTACTAC (SEQ ID NO: 26155) CAGTTTTCCATGGGTTCTACTAT (SEQID NO: 26156) TTATGAAATGGTACAGACAAGTGAT (SEQ ID NO: 26157) hCV1361979A/G CCAGTTTTGGTGTCAACTAGAAA (SEQ ID NO: 26158) CCAGTTTTGGTGTCAACTAGAAG(SEQ ID NO: 26159) TTGCAACCTGAAAAACATAACTA (SEQ ID NO: 26160) hCV1403468T/G ACTGGCCCCTTGCAT (SEQ ID NO: 26161) ACTGGCCCCTTGCAG (SEQ ID NO:26162) AGGAGGGAACCAAACCTTA (SEQ ID NO: 26163) hCV1487384 A/GACACCCACCCTACCACAA (SEQ ID NO: 26164) ACACCCACCCTACCACAG (SEQ ID NO:26165) TGTTAAACAGGAGGAAAGACAATA (SEQ ID NO: 26166) hCV1552900 A/GGGGATGGAAGAGCTTCA (SEQ ID NO: 26167) GGGATGGAAGAGCTTCG (SEQ ID NO:26168) CTGCAGCCTTCCTCTGAC (SEQ ID NO: 26169) hCV1575287 G/CCCAGGTGATCCAGGAGAG (SEQ ID NO: 26170) CCAGGTGATCCAGGAGAC (SEQ ID NO:26171) TCCATGGCGAAAATTTTG (SEQ ID NO: 26172) hCV15954277 A/GTGTCGGTAAACATGGCA (SEQ ID NO: 26173) GTCGGTAAACATGGCG (SEQ ID NO: 26174)GGTGGGTGGTCTGACTCTC (SEQ ID NO: 26175) hCV15963704 A/GAGATGGAGCTGCTCATCA (SEQ ID NO: 26176) GATGGAGCTGCTCATCG (SEQ ID NO:26177) GTTAGGAAGGTGATCCAAGATAC (SEQ ID NO: 26178) hCV1603656 G/AGCTGCCCTCAGTCCG (SEQ ID NO: 26179) TGCTGCCCTCAGTCCA (SEQ ID NO: 26180)GGGCACTGCCAATTCTTAG (SEQ ID NO: 26181) hCV1608777 G/ACAAGAGGATTTTTATGGAATGG (SEQ ID NO: 26182) ACAAGAGGATTTTTATGGAATGA (SEQID NO: 26183) GGTGGTGAGGCTTTGAGTATA (SEQ ID NO: 26184) hCV16165996 C/TCTGAGGCCTATGTCCTC (SEQ ID NO: 26185) CTGAGGCCTATGTCCTT (SEQ ID NO:26186) AGCTCTCCTTTGTTGCTACTG (SEQ ID NO: 26187) hCV16170982 C/GCCCCCACTCTCCAGC (SEQ ID NO: 26188) CCCCCACTCTCCAGG (SEQ ID NO: 26189)GGCAAAAGCACTGTGAAGA (SEQ ID NO: 26190) hCV16170993 G/A CGGAGCCTCCAGAAATG(SEQ ID NO: 26191) AACGGAGCCTCCAGAAATA (SEQ ID NO: 26192)GCAGCAGGCTCCACAGT (SEQ ID NO: 26193) hCV16172262 G/A GCCTCTCACCTTGAACG(SEQ ID NO: 26194) GGCCTCTCACCTTGAACA (SEQ ID NO: 26195)CACTTGGTCCCAGCACTACT (SEQ ID NO: 26196) hCV16173091 T/CCACAGACTTGATGTTTTTGAAAGT (SEQ ID NO: 26197) CAGACTTGATGTTTTTGAAAGC (SEQID NO: 26198) TGCAAAGGAAGCAACTTCA (SEQ ID NO: 26199) hCV16179628 T/CAGGTTCACTGTTTCTCCAAT (SEQ ID NO: 26200) AGGTTCACTGTTTCTCCAAC (SEQ ID NO:26201) GAGCACATCCTTCCATTGTAA (SEQ ID NO: 26202) hCV16181123 C/AAATTCAAGACAGTGCAACATC (SEQ ID NO: 26203) TAAATTCAAGACAGTGCAACATA (SEQ IDNO: 26204) CACACACCGCACTCTAATTACT (SEQ ID NO: 26205) hCV16195242 G/CCTTACAGAAGGACATCCTTTACAG (SEQ ID NO: 26206) CTTACAGAAGGACATCCTTTACAC(SEQ ID NO: 26207) GCAGGCCCAAGATTGAT (SEQ ID NO: 26208) hCV16266313 T/CCACGGCGCACTTTCTT (SEQ ID NO: 26209) CACGGCGCACTTTCTC (SEQ ID NO: 26210)TGTTTTTTCCTTTGTCATCTTATCTA (SEQ ID NO: 26211) hCV1639938 T/GAGTGGAGCTTCAGGGCT (SEQ ID NO: 26212) TGGAGCTTCAGGGCG (SEQ ID NO: 26213)CAGTGGAGACAGAGGATGTTTAC (SEQ ID NO: 26214) hCV1932478 T/CCAGCATTCCCATCACATT (SEQ ID NO: 26215) CAGCATTCCCATCACATC (SEQ ID NO:26216) TTTTGCTTTTGTGATTCTATTACTTA (SEQ ID NO: 26217) hCV1985480 T/CCAGCTGCTGCTGTCCAT (SEQ ID NO: 26218) CAGCTGCTGCTGTCCAC (SEQ ID NO:26219) CATGAACCTGTCAATCTTCTCA (SEQ ID NO: 26220) hCV2143203 C/TCAGATATGGACTCCAGTCATAGTAC (SEQ ID NO: 26221) CAGATATGGACTCCAGTCATAGTAT(SEQ ID NO: 26222) TGGCTGAGCATCGTTATTC (SEQ ID NO: 26223) hCV2143205 T/CTGTCGAATGGGAGTCTTCTT (SEQ ID NO: 26224) TGTCGAATGGGAGTCTTCTC (SEQ ID NO:26225) GGAAGAAACAGCTACCCAGA (SEQ ID NO: 26226) hCV22213764 C/TTCCTGCCTAGGACGC (SEQ ID NO: 26227) CTCCTGCCTAGGACGT (SEQ ID NO: 26228)GCACTCAGCCCATCAGATG (SEQ ID NO: 26229) hCV22271841 C/T CATCACGGAGATCCACC(SEQ ID NO: 26230) ATCATCACGGAGATCCACT (SEQ ID NO: 26231)TCAGCTCCAAGGAGATTCTTAG (SEQ ID NO: 26232) hCV22272408 A/GTGTCGGTAAACATGGCA (SEQ ID NO: 26233) GTCGGTAAACATGGCG (SEQ ID NO: 26234)GGTGGGTGGTCTGACTCTC (SEQ ID NO: 26235) hCV22272567 A/GTAGACAACGGGAAGATTATAGAGTA (SEQ ID NO: 26236) ACAACGGGAAGAATTAGAGTG (SEQID NO: 26237) TTTTGTGCTGTTCACATTCTC (SEQ ID NO: 26238) hCV22273419 C/TACTCCTTTGGACTGGC (SEQ ID NO: 26239) TGACTCCTTTGGACTGGT (SEQ ID NO:26240) TCTTAAATGCTGTGGAATTGTG (SEQ ID NO: 26241) hCV22274307 C/TGACCTACGCTCCTTCATCTAAC (SEQ ID NO: 26242) GACCTACGCTCCTTCATCTAAT (SEQ IDNO: 26243) CAATGTGATTACCTCAAAATAATATCTAC (SEQ ID NO: 26244) hCV22274624G/A CCCTACAGAGGATGTCAG (SEQ ID NO: 26245) CCCTACAGAGGATGTCAA (SEQ ID NO:26246) CAGAGCCTCCCTTGTCAC (SEQ ID NO: 26247) hCV22274712 G/CCTTACAGAAGGACATCCTTTACAG (SEQ ID NO: 26248) CTTACAGAAGGACATCCTTTACAC(SEQ ID NO: 26249) GCAGGCCCAAGATTGAT (SEQ ID NO: 26250) hCV2531086 A/GGCCCCCCTCTCTGAAGA (SEQ ID NO: 26251) CCCCCCTCTCTGAAGG (SEQ ID NO: 26252)CCAGTTCGTGGTATGTTCATCT (SEQ ID NO: 26253) hCV2531431 G/A GCCAATGTGGCGGG(SEQ ID NO: 26254) GCCAATGTGGCGGA (SEQ ID NO: 26255) CCTGGACGACGGTTTCA(SEQ ID NO: 26256) hCV25472673 C/T TGGGCTCCATCCCAC (SEQ ID NO: 26257)TGGGCTCCATCCCAT (SEQ ID NO: 26258) CCAATTCTTTTTCTTCTTTCAGTT (SEQ ID NO:26259) hCV25472687 G/T TTTGTACAGGAAGTTGTGAGG (SEQ ID NO: 26260)TGTTTGTACAGGAAGTTGTGAGT (SEQ ID NO: 26261) GGCTGTCTCGGCTTCTTT (SEQ IDNO: 26262) hCV25473867 T/C CGGATCAGGAAGGCTT (SEQ ID NO: 26263)CGGATCAGGAAGGCTC (SEQ ID NO: 26264) ATCCACAGACAGGCAATCTATA (SEQ ID NO:26265) hCV25474320 C/T ACTAGAGATGGATGAACTTTTCTC (SEQ ID NO: 26266)ACTAGAGATGGATGAACTTTTCTT (SEQ ID NO: 26267) GTTCTCCAGAAAGCATTCTTTAT (SEQID NO: 26268) hCV2548962 C/T CAAATACATCTCCCAGGATC (SEQ ID NO: 26269)CAAATACATCTCCCAGGATT (SEQ ID NO: 26270) GTTTTAATTGCAGTTTGAATGATAT (SEQID NO: 26271) hCV25591528 T/C TCCAAAAGGACCTGACAT (SEQ ID NO: 26272)TCCAAAAGGACCTGACAC (SEQ ID NO: 26273) GGCTGCAGAATGGAATTT (SEQ ID NO:26274) hCV25591743 T/A TGGTTGGTGTCAATCCTCT (SEQ ID NO: 26275)GGTTGGTGTCAATCCTCA (SEQ ID NO: 26276) CCAGGTCCCCAATCTACTCT (SEQ ID NO:26277) hCV25594815 G/A GCTGGGCCGTCTG (SEQ ID NO: 26278) AGCTGGGCCGTCTA(SEQ ID NO: 26279) CACCGCTCATGGACACA (SEQ ID NO: 26280) hCV25603879 C/TGAAGTCATTCTGCTCTGC (SEQ ID NO: 26281) ATGAAGTCATTCTGCTCTGT (SEQ ID NO:26282) TTTCCATCTCCTAACTCTTTTCTAG (SEQ ID NO: 26283) hCV25608809 G/AAGGGAAACCCCAGAGAG (SEQ ID NO: 26284) AGGGAAACCCCAGAGAA (SEQ ID NO:26285) ACAAGTTCATTTGTGAATGTGA (SEQ ID NO: 26286) hCV25608818 T/CTCAATGTCGTATTCACCTTCT (SEQ ID NO: 26287) AATGTCGTATTCACCTTCC (SEQ ID NO:26288) ACTCCAGGATTTTTCAAAGATTAT (SEQ ID NO: 26289) hCV25614474 A/GCTGTTGTCCTGCTTCCAA (SEQ ID NO: 26290) CTGTTGTCCTGCTTCCAG (SEQ ID NO:26291) GTTTCTGCATCAGTGAGATTTT (SEQ ID NO: 26292) hCV25627634 C/GCACCCTGCAGATGGAAC (SEQ ID NO: 26293) CACCCTGCAGATGGAAG (SEQ ID NO:26294) CAAGACTCCTTCATCCTCAATAGT (SEQ ID NO: 26295) hCV25629888 G/CGCACCTTGCTGCTGTCTG (SEQ ID NO: 26296) GCACCTTGCTGCTGTCTC (SEQ ID NO:26297) GCCCTGATCACTGCAAAAC (SEQ ID NO: 26298) hCV25638153 G/CCCTCTCCACAGCGTTTTG (SEQ ID NO: 26299) CCTCTCCACAGCGTTTTC (SEQ ID NO:26300) GCAGCGGCCACAGAG (SEQ ID NO: 26301) hCV25646316 G/CCCCCCACAGAAAGCG (SEQ ID NO: 26302) CCCCCACAGAAAGCC (SEQ ID NO: 26303)TCTAAAATCCCATCACAAACTTTA (SEQ ID NO: 26304) hCV2562767 A/GAGACAAGATTGAACGGATCA (SEQ ID NO: 26305) GACAAGATTGAACGGATCG (SEQ ID NO:26306) TGAAATCCTCAAACACAGACAC (SEQ ID NO: 26307) hCV25653599 C/TAAGAATTTGAACTTACTGATGAGAC (SEQ ID NO: 26308) TAAGAATTTGAACTTACTGATGAGATATGTGGAGTTTGATTTCCTTATTA (SEQ ID NO: 26310) (SEQ ID NO: 26309)hCV25944011 C/A GTATGACCTGTACTTCTGGAGAC (SEQ ID NO: 26311)GTATGACCTGTACTTCTGGAGAA (SEQ ID NO: 26312) AGGCCCCCTCTCAATACT (SEQ IDNO: 26313) hCV25963638 T/C TGCGAATCCCACTCTACTT (SEQ ID NO: 26314)TGCGAATCCCACTCTACTC (SEQ ID NO: 26315) CAAGGGAAAAGTGTCAATCAG (SEQ ID NO:26316) hCV2676030 A/G ACAATCACAAGCTATTTGTTCA (SEQ ID NO: 26317)CAATCACAAGCTATTTGTTCG (SEQ ID NO: 26318) AACTTTTGGTTTTGTTTGTTACTCT (SEQID NO: 26319) hCV2705229 T/C GAGCCGGAGCCTCT (SEQ ID NO: 26320)AGCCGGAGCCTCC (SEQ ID NO: 26321) GCATGCCTTCCTCACTAGAC (SEQ ID NO: 26322)hCV2782570 T/C CCAGCAAACTATGATGAATAAT (SEQ ID NO: 26323)CCAGCAAACTATGATGAATAAC (SEQ ID NO: 26324) TGGGATGACTCTAGCCACTTAC (SEQ IDNO: 26325) hCV2822674 T/C GATGCTGGGTGGATGTT (SEQ ID NO: 26326)GATGCTGGGTGGATGTC (SEQ ID NO: 26327) TGTGGCCCTGAGAATGTAC (SEQ ID NO:26328) hCV2908485 T/C CAAAGCCATGGTCTTTCAT (SEQ ID NO: 26329)CAAAGCCATGGTCTTTCAC (SEQ ID NO: 26330) CCGCTCTTCGGTTCTACTC (SEQ ID NO:26331) hCV3084793 C/T CCCGGCTGGGCGCGGACATGGAGGACGTTCCCCGGCTGGGCGCGGACATGGAGGACGTT CAGCTTGCGCAGGTG (SEQ ID NO: 26334) (SEQ IDNO: 26332) (SEQ ID NO: 26333) hCV3135085 C/A CTGGAAATGGTTATGGGC (SEQ IDNO: 26335) TACTGGAAATGGTTATGGGA (SEQ ID NO: 26336)TTTATAGGCGTGAAACTAATTCTC (SEQ ID NO: 26337) hCV313778 G/ATCATGCAGTCTCTCCTGAAG (SEQ ID NO: 26338) TCATGCAGTCTCTCCTGAAA (SEQ ID NO:26339) TCTGCCTTATCACTTGTACACTG (SEQ ID NO: 26430) hCV3216553 A/GGCCATACACCTCTTTCAGGA (SEQ ID NO: 26341) CCATACACCTCTTTCAGGG (SEQ ID NO:26342) CCAGGAGGCATGTTGATAAG (SEQ ID NO: 26343) hCV3230004 G/TGTAACACTGGGGACAACG (SEQ ID NO: 26344) GTGTAACACTGGGGACAACT (SEQ ID NO:26345) ACCAAGGAGAACAAGACACATA (SEQ ID NO: 26346) hCV342590 C/TAGACAAATTCTCTCATGTCCAC (SEQ ID NO: 26347) AGACAAATTCTCTCATGTCCAT (SEQ IDNO: 26348) TGTTTTTCCAGGAAAAAGATATTC (SEQ ID NO: 26349) hCV5478 G/ACGGCTTTCTGGTGGG (SEQ ID NO: 26350) ACGGCTTTCTGGTGGA (SEQ ID NO: 26351)GGCTCCGAGGACGAGA (SEQ ID NO: 26352) hCV5687 T/C CCCTCAGTGTGACTGAGAT (SEQID NO: 26353) CCCTCAGTGTGACTGAGAC (SEQ ID NO: 26354) CCAGGCATTTCCCATACAG(SEQ ID NO: 26355) hCV598677 G/T CCAAGCTGAAAGGCAAG (SEQ ID NO: 26356)CCAAGCTGAAAGGCAAT (SEQ ID NO: 26357) CAGCCAGGGTGGAGAGT (SEQ ID NO:26358) hCV7482175 A/G TGCCCAGGGCTCTGATA (SEQ ID NO: 26359)GCCCAGGGCTCTGATG (SEQ ID NO: 26360) CCATCGCATGCTCAATACA (SEQ ID NO:26361) hCV7490119 G/C GCCTTGGGGCACATG (SEQ ID NO: 26362) GCCTTGGGGCACATC(SEQ ID NO: 26363) GGAATTTATGGCAGTTTTAAACAT (SEQ ID NO: 26364)hCV7490135 G/A GCAGTCCTGAACAAAGTAGATG (SEQ ID NO: 26365)CGCAGTCCTGAACAAAGTAGATA (SEQ ID NO: 26366) CGTGCATGTTTTGAAAAATGTA (SEQID NO: 26367) hCV7530616 T/C GACATCAAACAAGCTCTTTCT (SEQ ID NO: 26368)ACATCAAACAAGCTCTTTCC (SEQ ID NO: 26369) AACATGGGACCCAACACTT (SEQ ID NO:26370) hCV7582933 C/T CCAAAGGGTGTCAAGGC (SEQ ID NO: 26371)TCCAAAGGGTGTCAAGGT (SEQ ID NO: 26372) GCTGCTGGAATATGTTTGAGA (SEQ ID NO:26373) hCV7584364 G/A AGGAACAGCAAGAACCG (SEQ ID NO: 26374)CAGGAACAGCAAGAACCA (SEQ ID NO: 26375) CAGGAGCCTGCACTCTG (SEQ ID NO:26376) hCV761961 G/A CACAGTCAAAGAATCAAGCG (SEQ ID NO: 26377)TCACAGTCAAAGAATCAAGCA (SEQ ID NO: 26378) CCGTTTGAATTTTCCAATAAG (SEQ IDNO: 26379) hCV783138 G/A ATGCCACCCACTACCG (SEQ ID NO: 26380)CATGCCACCCACTACCA (SEQ ID NO: 26381) TTTTGCAGCCACTCGAT (SEQ ID NO:26382) hCV7900503 C/T CGTCTCCAGGAAAATCATAAC (SEQ ID NO: 26383)CGTCTCCAGGAAAATCATAAT (SEQ ID NO: 26384) TGAGTTATTGCTACTTCAGAATCAT (SEQID NO: 26385) hCV790057 A/G AGCAGCTCCGAGTCCA (SEQ ID NO: 26386)AGCAGCTCCGAGTCCG (SEQ ID NO: 26387) GGCCCACAAGGTGAAAT (SEQ ID NO: 26388)hCV8705506 C/G CCACTTCGGGTTCCTC (SEQ ID NO: 26389) CCACTTCGGGTTCCTG (SEQID NO: 26390) CCCTGGCTTCAACATGA (SEQ ID NO: 26391) hCV8827241 G/CTCAAGAGGACAGTGATGGTG (SEQ ID NO: 26392) TCAAGAGGACAGTGATGGTC (SEQ ID NO:26393) TGGTTAGAATCTGTGAAGGAACTA (SEQ ID NO: 26394) hCV8921288 G/TCCGCAGAGGTGTGGG (SEQ ID NO: 26395) CCGCAGAGGTGTGGT (SEQ ID NO: 26396)CATTTTGCGGTGGAAATG (SEQ ID NO: 26397) hCV8932279 A/GGAGTGGCCCTATCAAATGTTA (SEQ ID NO: 26398) GTGGCCCTATCAAATGTTG (SEQ ID NO:26399) TTTGTTGTGCCTGATGATGTA (SEQ ID NO: 26400) hCV9458082 C/TACAGTCAGGTGGATCTCC (SEQ ID NO: 26401) CACAGTCAGGTGGATCTCT (SEQ ID NO:26402) CACATTCCTGGAGGTGCTAG (SEQ ID NO: 26403) hCV9482394 G/ACGCGCGCTAACCG (SEQ ID NO: 26404) CGCGCGCTAACCA (SEQ ID NO: 26405)CGTCCGTCATGGATCAGA (SEQ ID NO: 26406) hCV9506149 T/A CTGCTGGCCGTCCT (SEQID NO: 26407) TGCTGGCCGTCCA (SEQ ID NO: 26408) ACTCACGCTTGCTTTGACT (SEQID NO: 26409) hCV9578831 A/T TTAAATAAGGCTCTACCTCAAAGA (SEQ ID NO: 26410)TTAAATAAGGCTCTACCTCAAAGT (SEQ ID NO: 26411) TTTTGATGCCTGAGAAAGAAG (SEQID NO: 26412) hCV9698625 T/C TTTGCAGTTTATTAAGATGAGGAT (SEQ ID NO: 26413)TTTGCAGTTTATTAAGATGAGGAC (SEQ ID NO: 26414) CGGTGCTCCCTGTCATAA (SEQ ISNO: 26415)

TABLE 6 Gene Case Cntrl Marker Name Sample Set p-value OR 95% Cl Freq.Freq. Allele1 Mode Strata Adjust hCV1608777 PLSCR1 Sample Set 2 0.03440.51 0.27-0.96 5.1 9.6 T Rec Age T2 hCV1608777 PLSCR1 Sample Set 10.0076 0.19 0.05-0.72 2.8 13.2 T Rec Age T2 hCV16165996 LRP2 Sample Set2 0.0306 1.31 1.03-1.68 49.0 42.3 T Dom ALL hCV16165996 LRP2 Sample Set2 0.0281 1.31 1.02-1.68 28.3 23.2 T Add Smoke+ hCV16165996 LRP2 SampleSet 1 0.0037 1.69 1.18-2.4  47.9 35.2 T Dom ALL hCV16165996 LRP2 SampleSet 1 0.0321 1.67 1.04-2.7  48.3 35.9 T Dom Smoke+ hCV16181123 ACADLSample Set 2 0.0182 1.74 1.09-2.77 19.3 12.1 G Rec MI− hCV16181123 ACADLSample Set 1 0.0072 1.87 1.18-2.96 66.9 52.0 G Dom MI− hCV2143203 CD44Sample Set 2 0.0394 4.00 0.97- 6.7 1.8 T Rec Age T1 16.49 hCV2143203CD44 Sample Set 2 0.0326 2.08 1.05-4.11 26.0 14.5 T Add MI−Age T1hCV2143203 CD44 Sample Set 1 0.0274 6.00  0.7-50.8 5.5 0.0 T Rec Age T1hCV2143203 CD44 Sample Set 1 0.0163 8.90  0.8-88.9 6.7 0.0 T Rec MI−AgeT1 hCV2143205 CD44 Sample Set 2 0.0090 1.68 1.12-2.52 39.8 28.3 A AddMI−Smoke− hCV2143205 CD44 Sample Set 1 0.0123 3.88 1.3-1.2 18.2 5.4 ARec MI−Smoke− hCV22273419 GP6 Sample Set 2 0.0332 0.81 0.67-0.99 15.518.4 C Add ALL hCV22273419 GP6 Sample Set 1 0.0229 0.51 0.29-0.92 27.742.7 C Dom Smoke− hCV25591528 CD163 Sample Set 2 0.0285 0.73 0.55-0.979.7 12.8 A Add ALL hCV25591528 CD163 Sample Set 2 0.0356 0.20   0-1.50.0 1.9 A Rec Male hCV25591528 CD163 Sample Set 2 0.0105 0.51  0.3-0.887.3 13.4 A Add Smoke− hCV25591528 CD163 Sample Set 1 0.0096 0.10 0-1 0.02.2 A Rec ALL hCV25591528 CD163 Sample Set 1 0.0328 0.20   0-1.4 0.0 2.5A Rec Male hCV25591528 CD163 Sample Set 1 0.0457 0.52 0.28-0.99 7.8 14.0A Allelic Smoke− hCV25603879 SCNN1A Sample Set 2 0.0330 2.24 1.05-4.7611.1 5.3 T Dom Smoke− hCV25603879 SCNN1A Sample Set 2 0.0243 2.711.08-6.8  6.7 2.6 T Add MI−Age T1 hCV25603879 SCNN1A Sample Set 2 0.04291.65 1.01-2.68 9.5 6.0 T Dom MI− hCV25603879 SCNN1A Sample Set 2 0.01002.26 1.18-4.32 4.8 2.2 T Add MI−Male hCV25603879 SCNN1A Sample Set 20.0474 1.83   1-3.33 10.4 6.0 T Dom MI− hCV25603879 SCNN1A Sample Set 20.0054 3.22 1.36-7.62 15.3 5.3 T Dom MI−Smoke− hCV25603879 SCNN1A SampleSet 1 0.0147 3.67  1.2-11.5 6.4 1.8 T Add Smoke− hCV25603879 SCNN1ASample Set 1 0.0201 5.99 1.07-33.6 6.7 1.2 T Add MI−Age T1 hCV25603879SCNN1A Sample Set 1 0.0035 3.62 1.43-9.2  5.4 1.6 T Add MI− hCV25603879SCNN1A Sample Set 1 0.0165 3.93  1.2-13.3 6.3 1.7 T Add MI−MalehCV25603879 SCNN1A Sample Set 1 0.0035 3.62 1.43-9.2  5.4 1.6 T Add MI−hCV25603879 SCNN1A Sample Set 1 0.0020 5.53  1.6-18.9 9.3 1.8 T AddMI−Smoke− hCV25627634 SMTN Sample Set 2 0.0396 1.82 1.02-3.24 64.4 49.8G Dom MI−Smoke− hCV25627634 SMTN Sample Set 1 0.0068 2.62 1.28-5.4  15.76.6 G Rec MI− hCV3084793 APOE Sample Set 2 0.0487 1.62   1-2.64 30.521.3 C Dom MI−Female hCV3084793 APOE Sample Set 2 0.0299 1.82 1.06-3.1438.5 25.6 C Dom MI−Age T2 hCV3084793 APOE Sample Set 1 0.0069 2.251.2-4.2 23.2 11.8 C Add MI−Female hCV3084793 APOE Sample Set 1 0.01378.40  0.9-78.2 7.9 0.0 C Rec MI−Age T2 hCV5478 TCF1 Sample Set 2 0.01003.07 1.24-7.57 3.5 1.3 T Allelic Age T2 hCV5478 TCF1 Sample Set 1 0.015212.21 1.18-   4.6 0.6 T Allelic Age T2 126.50 hCV598677 EDN1 Sample Set2 0.0065 1.82 1.18-2.79 45.8 31.7 T Dom Age T1 hCV598677 EDN1 Sample Set2 0.0048 3.23 1.38-7.53 60.0 31.7 T Dom MI−Age T1 hCV598677 EDN1 SampleSet 1 0.0372 4.60 0.97-22   9.9 2.3 T Rec Age T1 hCV598677 EDN1 SampleSet 1 0.0048 8.31  1.5-45.4 16.7 2.4 T Rec MI−Age T1 hCV7482175 HLA-ASample Set 2 0.0163 0.66 0.47-0.93 25.7 34.4 A Dom Smoke+ hCV7482175HLA-A Sample Set 1 0.0132 0.61 0.4-0.9 14.3 21.6 A Add Smoke+ hCV7530616WRN Sample Set 2 0.0027 14.10 1.46-   1.9 0.1 A Rec MI− 136.45hCV7530616 WRN Sample Set 1 0.0492 5.90  0.5-57.9 3.6 0.0 A RecMI−Female hCV7530616 WRN Sample Set 1 0.0230 2.43 1.1-5.2 11.7 5.2 A AddMI−Smoke+ hCV7584364 PTGS1 Sample Set 2 0.0006 9.32 1.97- 2.7 0.3 T RecMI− 44.18 hCV7584364 PTGS1 Sample Set 2 0.0051 11.90  1.2-117 3.6 0.0 TRec MI−Age T1 hCV7584364 PTGS1 Sample Set 1 0.0209 1.99 1.10-3.6  10.05.3 T Add MI− hCV7584364 PTGS1 Sample Set 1 0.0282 3.11 1.1-8.7 13.3 4.7T Add MI−Age T1 hCV761961 FABP2 Sample Set 2 0.0360 1.48 1.03-2.14 50.040.3 T Dom Age T3 hCV761961 FABP2 Sample Set 1 0.0397 6.73 0.84-53.9 9.51.5 T Rec Age T3 hCV790057 CETP Sample Set 2 0.0121 0.78 0.64-0.95 26.331.4 G Allelic ALL hCV790057 CETP Sample Set 2 0.0443 0.61 0.37-0.99 6.911.0 G Rec MI− hCV790057 CETP Sample Set 1 0.0033 0.27 0.11-0.67 30.061.2 G Dom MI−Age T1 hCV790057 CETP Sample Set 1 0.0186 0.49 0.27-0.8944.0 61.6 G Dom Age T1 hCV790057 CETP Sample Set 1 0.0482 0.49 0.24-1.0 37.2 55.0 G Dom MI−Smoke− hCV8705506 KLK1 Sample Set 2 0.0122 0.580.38-0.89 45.6 59.2 C Dom Smoke− hCV8705506 KLK1 Sample Set 1 0.03880.57 0.34-0.98 9.2 15.0 C Rec ALL hCV8921288 GAPD Sample Set 2 0.01301.38 1.7-1.77 40.9 33.5 G Dom ALL hCV8921288 GAPD Sample Set 1 0.04866.45 0.78-53.3 5.8 1.0 G Rec Female hCV9458082 NOS2A Sample Set 2 0.00921.47  1.1-1.96 17.8 12.9 T Rec ALL hCV9458082 NOS2A Sample Set 1 0.04061.87 1.02-3.4  66.4 51.3 T Dom Age T2 hCV9458082 NOS2A Sample Set 10.0200 1.49 1.06-2.1  41.3 32.1 T Add Smoke+ hCV9458082 NOS2A Sample Set1 0.0228 1.74 1.08-2.8  68.0 55.0 T Dom Male hCV9506149 FN1 Sample Set 20.0058 1.32 1.08-1.6  28.7 23.4 T Add ALL hCV9506149 FN1 Sample Set 10.0152 1.60 1.08-2.4  27.3 19.0 T Add Male hCV1552900 ALOX12 Sample Set2 0.1180 1.38 0.92-2.06 50.0 42.1 A Allelic Smoke− gender,age_group_lt54_le64 hCV1552900 ALOX12 Sample Set 1 0.2066 1.40 0.83-2.3350.0 42.5 A Allelic Smoke− gender, age_group_lt54_le64 hCV1552900 ALOX12Meta 0.0455 1.38 1.01-1.90 50.0 42.2 A Allelic Smoke− source, gender,age_group_lt54_le64 hCV16266313 BIRC5 Sample Set 2 0.3686 1.37 0.69-2.726.1 4.9 G Allelic Age T3 gender, smoke hCV16266313 BIRC5 Sample Set 10.0280 3.21 1.08-9.60 9.5 3.1 G Allelic Age T3 gender, smoke hCV16266313BIRC5 Meta 0.0359 1.83 1.04-3.22 7.7 4.5 G Allelic Age T3 source,gender, smoke hCV1985480 AGT Sample Set 2 0.2513 0.84 0.63-1.13 11.814.2 A Allelic ALL gender, age_group_lt54_le64, smoke hCV1985480 AGTSample Set 1 0.0447 0.57 0.32-0.99 7.5 12.1 A Allelic ALL gender,age_group_lt54_le64, smoke hCV1985480 AGT Meta 0.0478 0.77 0.59-1.0010.6 13.7 A Allelic ALL source, gender, age_group_lt54_le64, smokehCV22274624 HDLBP Sample Set 2 0.0348 0.67 0.47-0.97 40.1 48.7 C Dom ALLgender, age_group_lt54_le64 hCV22274624 HDLBP Sample Set 1 0.4815 0.850.54-1.34 38.8 43.3 C Dom ALL gender, age_group_lt54_le64 hCV22274624HDLBP Meta 0.0369 0.74 0.55-0.98 39.6 47.4 C Dom ALL source, gender,age_group_lt54_le64 hCV2548962 PON1 Sample Set 2 0.1393 0.85 0.69-1.0527.2 30.4 C Allelic Smoke+ gender, age_group_lt54_le64, smoke hCV2548962PON1 Sample Set 1 0.0545 0.71 0.50-1.01 28.1 35.9 C Allelic Smoke+gender, age_group_lt54_le64, smoke hCV2548962 PON1 Meta 0.0238 0.810.68-0.97 27.4 31.7 C Allelic Smoke+ source, gender,age_group_lt54_le64, smoke hCV11660791 MTHFD1 Sample Set 2 0.01 1.51.1-2.1 88 81 C Allelic ALL hCV11660791 MTHFD1 Sample Set 1 0.0364 0.580.3-1.0 14 21 T Allelic Age T3

TABLE 7 Marker Gene Name Sample Set p-value OR 95% Cl Case Freq. CntrlFreq. Allele1 Mode Strata hCV11975277 SELP Sample Set 1 0.039 n/a* G RecAge T3 hCV1575287 IL8RA Sample Set 1 0.027 n/a* G Dom Older hCV1575287IL8RA Sample Set 1 0.046 n/a* G Dom Smoke− hCV1603656 HSPG2 Sample Set 10.037 1.13 T Allelic Female hCV1639938 F13A1 Sample Set 1 0.04 n/a* ADom Age T2 hCV3216553 APOB Sample Set 1 0.047 n/a* A Dom Age T2hCV3216553 APOB Sample Set 1 0.034 1.55 A Allelic Age T1 hCV7582933PLA2G7 Sample Set 1 0.019 5.95 T Rec Age T3 hCV1129436 APOL3 Sample Set1 0.02463 C Allelic Smoke+ hCV1129436 APOL3 Sample Set 1 0.02409 CAllelic male/age T2 hCV1129436 APOL3 Sample Set 1 0.02811 C Allelicmale/ no hypertension hCV1129436 APOL3 Sample Set 1 0.00455 C AllelicMaleSmoke+ hCV11623862 TBXAS1 Sample Set 1 0.04724 T Allelic AllhCV11972321 F13A1 Sample Set 1 0.04190 G Allelic Smoke+ hCV11972321F13A1 Sample Set 1 0.04755 G Allelic female/ no hypertension hCV11972321F13A1 Sample Set 1 0.02064 G Allelic male/age T1 hCV11972321 F13A1Sample Set 1 0.01487 G Allelic MaleSmoke+ hCV1202883 MTHFR Sample Set 10.01357 A Allelic MI+ hCV1202883 MTHFR Sample Set 1 0.03654 A AllelicMaleMI+ hCV1345898 CHUK Sample Set 1 0.02992 C Allelic male/age T3hCV15954277 PRKCQ Sample Set 1 0.03260 A Allelic female/ no hypertensionhCV15963704 ITGA3 Sample Set 1 0.03398 A Allelic HypertensionhCV15963704 ITGA3 Sample Set 1 0.02997 A Allelic male/hypertensionhCV16170982 SREBF2 Sample Set 1 0.04406 C Allelic female/age T2hCV16170982 SREBF2 Sample Set 1 0.04529 C Allelic FemaleMI− hCV16170993SELPLG Sample Set 1 0.04733 G Allelic Smoke+ hCV16172262 FABP6 SampleSet 1 0.04577 G Allelic MI− hCV16172262 FABP6 Sample Set 1 0.02549 GAllelic No hypertension hCV16172262 FABP6 Sample Set 1 0.04855 G AllelicMaleMI− hCV16172262 FABP6 Sample Set 1 0.03227 G Allelic male/ nohypertension hCV16179628 ABCC2 Sample Set 1 0.03322 T Allelic female/ageT2 hCV16195242 MTP Sample Set 1 0.00814 G Allelic MI− hCV16195242 MTPSample Set 1 0.01728 G Allelic Smoke+ hCV16195242 MTP Sample Set 10.00212 G Allelic MaleMI− hCV16195242 MTP Sample Set 1 0.02587 G AllelicMaleSmoke+ hCV1932478 P2RY12 Sample Set 1 0.04012 T Allelic MI−hCV2213764 MMP11 Sample Set 1 0.02493 C Allelic Smoke+ hCV2213764 MMP11Sample Set 1 0.02596 C Allelic FemaleMI+ hCV22271841 PDGFRA Sample Set 10.04281 C Allelic MI+ hCV22271841 PDGFRA Sample Set 1 0.03142 C Allelicfemale/ hypertension hCV22272408 PRKCQ Sample Set 1 0.02252 A Allelicfemale/ no hypertension hCV22272408 PRKCQ Sample Set 1 0.04503 A AllelicMaleSmoke− hCV22272567 ABCC2 Sample Set 1 0.03614 A Allelic MI−hCV22272567 ABCC2 Sample Set 1 0.01204 A Allelic MaleMI− hCV22272567ABCC2 Sample Set 1 0.01309 A Allelic male/hypertension hCV22274307 MTPSample Set 1 0.03426 C Allelic age T2 hCV22274307 MTP Sample Set 10.04299 C Allelic MI− hCV22274307 MTP Sample Set 1 0.02673 C Allelicmale/age T2 hCV25472673 NPC1 Sample Set 1 0.02505 C Allelic MI+hCV25591743 ABCC2 Sample Set 1 0.02414 T Allelic MI− hCV25591743 ABCC2Sample Set 1 0.03752 T Allelic male/hypertension hCV25614474 PLG SampleSet 1 0.01528 A Allelic female hCV25614474 PLG Sample Set 1 0.02338 AAllelic MI− hCV25614474 PLG Sample Set 1 0.00122 A Allelic FemaleMI−hCV25614474 PLG Sample Set 1 0.02906 A Allelic female/ hypertensionhCV25629888 TIMP2 Sample Set 1 0.01835 G Allelic age T1 hCV25638153APOA5 Sample Set 1 0.03379 G Allelic age T3 hCV25646316 LRP2 Sample Set1 0.04595 G Allelic male/ no hypertension hCV25652767 LRP1 Sample Set 10.02443 A Allelic MI+ hCV25652767 LRP1 Sample Set 1 0.00243 A AllelicMI− hCV2705229 ITGA10 Sample Set 1 0.03914 T Allelic HypertensionhCV2705229 ITGA10 Sample Set 1 0.00468 T Allelic male/hypertensionhCV2822674 CUBN Sample Set 1 0.02925 T Allelic female hCV2822674 CUBNSample Set 1 0.00076 T Allelic female/age T3 hCV2822674 CUBN Sample Set1 0.03335 T Allelic FemaleMI+ hCV3135085 CUBN Sample Set 1 0.00245 TAllelic No hypertension hCV3135085 CUBN Sample Set 1 0.01122 T Allelicfemale/ no hypertension hCV3135085 CUBN Sample Set 1 0.02797 T Allelicmale/age T3 hCV342590 F5 Sample Set 1 0.04203 C Allelic FemaleSmoke−hCV342590 F5 Sample Set 1 0.03999 C Allelic MaleSmoke+ hCV5687 CX3CR1Sample Set 1 0.03556 T Allelic male hCV7490135 NPC1 Sample Set 1 0.01191G Allelic age T2 hCV7490135 NPC1 Sample Set 1 0.04298 G Allelic MI+hCV7490135 NPC1 Sample Set 1 0.02428 G Allelic male/age T2 hCV7900503CX3CR1 Sample Set 1 0.02118 C Allelic female/age T1 hCV7900503 CX3CR1Sample Set 1 0.03188 C Allelic FemaleSmoke+ hCV7900503 CX3CR1 Sample Set1 0.02491 C Allelic MaleMI+ hCV1361979 ACAT2 Sample Set 2 0.0027 1.713881.2-2.4 0.7342 0.6171 A Dom Age T2 hCV1361979 ACAT2 Sample Set 2 0.02981.37325   1-1.8 0.7114 0.6422 A Dom Male hCV1361979 ACAT2 Sample Set 20.0152 1.2601  1-1.5 0.4429 0.3869 A Add Smoke+ hCV1361979 ACAT2 SampleSet 2 0.198 1.1936   1-1.4 0.4431 0.4 A Add ALL hCV1361979 ACAT2 SampleSet 2 0.00753 1.73944 1.2-2.6 0.7371 0.6171 A Dom Age T2 hCV1361979ACAT2 Sample Set 2 0.0301 1.47134   1-2.1 0.7253 0.6422 A Dom MalehCV1361979 ACAT2 Sample Set 2 0.0181 1.49327 1.1-2.1 0.7153 0.6272 A DomSmoke+ hCV1361979 ACAT2 Sample Set 2 0.0358 1.32885   1-1.7 0.7 0.6371 ADom ALL hCV1361979 ACAT2 Sample Set 2 0.0126 2.1026 1.2-3.8 0.77220.6171 A Dom MI−AgeT2 hCV1361979 ACAT2 Sample Set 2 0.00369 1.94091.2-3   0.7578 0.6171 A Dom MI−AgeT2 hCV1361979 ACAT2 Sample Set 20.0373 1.2953   1-1.7 0.4497 0.3869 A Add MI−Smoke+ hCV22274712 MTPSample Set 2 0.0403 1.41589 1-2 0.6321 0.5482 G Dom Age T2 hCV22274712MTP Sample Set 2 0.0369 1.43566 1-2 0.6596 0.5745 G Dom FemalehCV22274712 MTP Sample Set 2 0.0498 1.4498   1-2.1 0.6733 0.5871 G DomMI− hCV2531431 THBD Sample Set 2 0.0111 0.22613 0.1-0.8 0.0149 0.0625 TRec Age T3 hCV2531431 THBD Sample Set 2 0.0299 0.42323 0.2-0.9 0.02020.0464 T Rec Male hCV2531431 THBD Sample Set 2 0.00295 0.5156 0.3-0.80.1186 0.207 T Allelic Age T3 hCV25608818 SLC10A2 Sample Set 2 0.007376.0214 1.3-27  0.021 0.0035 A Add Female hCV25608818 SLC10A2 Sample Set2 0.0363 3.5574  1.1-11.6 0.022 0.0063 A Add Smoke− hCV25608818 SLC10A2Sample Set 2 0.0212 5.3694  1.1-26.8 0.0188 0.0035 A Add FemalehCV25608818 SLC10A2 Sample Set 2 0.0263 4.208 1.2-15  0.0259 0.0063 AAllelic Smoke− hCV25608818 SLC10A2 Sample Set 2 0.0101 7.4602  1.2-45.20.0259 0.0035 A Add MI−Female hCV25608818 SLC10A2 Sample Set 2 0.0341 —— 0.0104 0 A Rec MI−Male hCV25608818 SLC10A2 Sample Set 2 0.0156 5.5614 1.4-22.6 0.0339 0.0063 A Add MI−Smoke− hCV25608818 SLC10A2 Sample Set 20.0314 — — 0.0065 0 A Rec MI− hCV25608818 SLC10A2 Sample Set 2 0.001328.5895  1.8-41.7 0.0297 0.0035 A Add MI−Female hCV25608818 SLC10A2Sample Set 2 0.0239 4.3227  1.2-15.5 0.0265 0.0063 A Allelic MI−Smoke−hCV25653599 WDR12 Sample Set 2 0.00488 1.77148 1.2-2.6 0.2868 0.185 CDom Age T2 hCV25653599 WDR12 Sample Set 2 0.0125 1.4324 1.1-1.9 0.1480.1081 C Add Male hCV25653599 WDR12 Sample Set 2 0.0011 2.06754 1.3-3.20.3194 0.185 C Dom Age T2 hCV25653599 WDR12 Sampel Set 2 0.00925 1.54121.1-2.1 0.1574 0.1081 C Add Male hCV25653599 WDR12 Sample Set 2 0.04491.33735   1-1.8 0.2716 0.218 C Dom ALL hCV25653599 WDR12 Sample Set 20.00797 2.1608 1.2-3.8 0.3291 0.185 C Dom MI−AgeT2 hCV25653599 WDR12Sample Set 2 0.00341 2.0357 1.3-3.3 0.3438 0.2047 C Dom MI−MalehCV25653599 WDR12 Sample Set 2 0.0204 1.5756 1.1-2.3 0.3052 0.218 C DomMI− hCV25653599 WDR12 Sample Set 2 0.00334 1.6595 1.2-2.3 0.1675 0.1081C Add MI−Male hCV25653599 WDR12 Sample Set 2 0.0106 1.4203 1.1-1.90.1571 0.116 C Add MI− hCV25653599 WDR12 Sample Set 2 0.0257 1.71711.1-2.8 0.2805 0.185 C Dom MI−AgeT2 hCV25653599 WDR12 Sample Set 20.0289 1.4654   1-2.1 0.1559 0.112 C Add MI−Smoke+ hCV25963638 SRPXSample Set 2 0.0333 1.51   1-2.2 0.079 0.0538 A Allelic Smoke+hCV25963638 SRPX Sample Set 2 0.0246 1.9735 1.1-3.5 0.1392 0.0758 AAllelic MI−AgeT2 hCV25963638 SRPX Sample Set 2 0.00997 2.0235 1.2-3.40.1237 0.0652 A Allelic MI−Male hCV25963638 SRPX Sample Set 2 0.04651.8005   1-3.2 0.0928 0.0538 A Allelic MI−Smoke+ hCV25963638 SRPX SampleSet 2 0.048 1.9943 1-4 0.0769 0.0401 A Rec MI− hCV25963638 SRPX SampleSet 2 0.00373 1.8731 1.2-2.8 0.1156 0.0652 A Allelic MI−Male hCV25963638SRPX Sample Set 2 0.00569 1.893 1.2-3   0.0971 0.0538 A AllelicMI−Smoke+ hCV25963638 SRPX Sample Set 2 0.0174 1.9469 1.1-3.4 0.07520.0401 A Rec MI− hCV25963638 SRPX Sample Set 2 0.0204 1.7792 1.1-2.90.1273 0.0758 A Allelic MI−AgeT2 hCV2782570 PTGIS Sample Set 2 0.02020.7282 0.6-0.9 0.3184 0.3908 G Allelic Smoke− hCV2782570 PTGIS SampleSet 2 0.0439 0.3075 0.1-1   0.0526 0.153 G Rec MI−Female hCV2782570PTGIS Sample Set 2 0.0345 1.4814   1-2.1 0.697 0.6082 G Dom MI−Smoke+hCV2782570 PTGIS Sample Set 2 0.0428 0.4656 0.2-1   0.0776 0.153 G RecMI−Female hCV2908485 none Sample Set 2 0.00678 0.6959 0.5-0.9 0.36210.4492 G Add Age T3 hCV2908485 none Sample Set 2 0.00146 0.63584 0.5-0.80.5955 0.6984 G Dom Male hCV2908485 none Sample Set 2 0.00761 0.77510.6-0.9 0.3935 0.4557 G Allelic Smoke+ hCV2908485 none Sample Set 20.015 0.76396 0.6-0.9 0.6211 0.6821 G Dom ALL hCV2908485 none Sample Set2 0.00553 0.6407 0.5-0.9 0.3432 0.4492 G Add Age T3 hCV2908485 noneSample Set 2 0.00236 0.59936 0.4-0.8 0.5812 0.6984 G Dom Male hCV2908485none Sample Set 2 0.0142 0.66644 0.5-0.9 0.6187 0.7089 G Dom Smoke+hCV2908485 none Sample Set 2 0.0181 0.73421 0.6-0.9 0.6117 0.6821 G DomALL hCV2908485 none Sample Set 2 0.0473 1.9907 1-4 0.2414 0.1378 G RecMI−Female hCV2908485 none Sample Set 2 0.0102 0.5553 0.4-0.9 0.56250.6984 G Dom MI−Male hCV2908485 none Sample Set 2 0.00378 2.5953 1.3-5  0.2712 0.1254 G Rec MI−Smoke+ hCV2908485 none Sample Set 2 0.009780.6506 0.5-0.9 0.3526 0.4557 G Add MI−Smoke+ hCV2908485 none Sample Set2 0.0432 0.6917 0.5-1   0.5974 0.6821 G Dom MI− hCV2908485 none SampleSet 2 0.00684 0.6135 0.4-0.9 0.599 0.7089 G Dom MI−Smoke+ hCV2908485none Sample Set 2 0.007 0.6185 0.4-0.9 0.5888 0.6984 G Dom MI−MalehCV2908485 none Sample Set 2 0.00798 0.69 0.5-0.9 0.5968 0.6821 G DomMI− hCV2908485 none Sample Set 2 0.0178 0.5526 0.3-0.9 0.5761 0.7109 GDom MI−AgeT3 hCV2908485 none Sample Set 2 0.0431 1.7825   1-3.1 0.20350.1254 G Rec MI−Smoke− hCV783138 F7 Sample Set 2 0.0245 0 — 0 0.0177 ARec Female hCV11592758 BDNF Sample Set 2 0.003689 1.418521 0.857360730.8090617 C Allelic ALL hCV11592758 BDNF Sample Set 2 0.015578 1.4425730.85240696 0.8001411 C Allelic Male hCV11592758 BDNF Sample Set 20.048839 1.410359 0.85028022 0.8010637 C Allelic Younger hCV2531086 CD22Sample Set 2 0.016484 1.45531 0.80197937 0.7356524 G Allelic YoungerhCV25474320 LCP1 Sample Set 2 0.003797 0.421814 0.04477473 0.1000101 TAllelic Female hCV25474320 LCP1 Sample Set 2 0.040595 0.6094570.05770926 0.0913129 T Allelic Older hCV25608809 TLR5 Sample Set 20.033066 1.352714 0.58624226 0.5115828 A Allelic Female hCV25608809 TLR5Sample Set 2 0.001546 1.482334 0.59272859 0.4954267 A Allelic OlderhCV25608809 TLR5 Sample Set 2 0.037258 1.249806 0.56389988 0.5085033 AAllelic Smoke+ hCV2676030 EDG2 Sample Set 2 0.047657 1.202427 0.353895620.3129634 G Allelic ALL hCV2676030 EDG2 Sample Set 2 0.047791 1.2502910.359831 0.310138 G Allelic Smoke+ hCV7490119 NPC1 Sample Set 2 8.66E−050.684841 0.27083028 0.3516384 C Allelic ALL hCV7490119 NPC1 Sample Set 20.020383 0.703492 0.28775573 0.3647954 C Allelic Female hCV7490119 NPC1Sample Set 2 0.001487 0.672101 0.25987035 0.3431481 C Allelic MalehCV7490119 NPC1 Sample Set 2 0.000415 0.502755 0.23248743 0.3759751 CAllelic Smoke− hCV7490119 NPC1 Sample Set 2 0.005119 0.690612 0.288474030.3699039 C Allelic Older hCV7490119 NPC1 Sample Set 2 0.002153 0.6408580.24675175 0.3382584 C Allelic Younger hCV7490119 NPC1 Sample Set 20.039905 0.787741 0.28126535 0.3318993 C Allelic Smoke+ hCV8827241SPARCL1 Sample Set 2 0.011283 0.716295 0.57830575 0.6568944 C AllelicYounger hCV8932279 SERPINB5 Sample Set 2 0.026358 1.465232 0.488572580.3946682 G Allelic Smoke− hCV9482394 KIAA1608 Sample Set 2 0.043011.74935 0.12036092 0.0725434 A Allelic Smoke− hCV9482394 KIAA1608 SampleSet 2 0.02777 0.641196 0.06323883 0.0952556 A Allelic Smoke+ hCV9578831TNFRSF6 Sample Set 2 0.034212 0.615467 0.07409659 0.115064 T AllelicYounger hCV1309246 PPP1R12A Sample Set 2 0.03147 0.339874 0.012289950.0353173 C Allelic Male hCV1309246 PPP1R12A Sample Set 2 0.0323890.187827 0.00861413 0.0442151 C Allelic Smoke− hCV1403468 IL12A SampleSet 2 0.032773 0.663131 0.13769986 0.1940753 G Allelic Male hCV1487384CD33 Sample Set 2 0.008942 1.597195 0.83948062 0.7660464 G Allelic MalehCV16173091 FABP1 Sample Set 2 0.04212 0.66867 0.27076982 0.3570351 CAllelic Female hCV16173091 FABP1 Sample Set 2 0.040581 0.7384590.29386913 0.3604351 C Allelic Smoke+ hCV25594815 LRP3 Sample Set 20.003239 1.690589 0.14376135 0.0903416 A Allelic ALL hCV25594815 LRP3Sample Set 2 0.002488 1.960164 0.16132547 0.0893639 A Allelic MalehCV25594815 LRP3 Sample Set 2 0.019092 1.672316 0.14861088 0.094512 AAllelic Smoke+ hCV25944011 EGLN2 Sample Set 2 0.047039 1.2645560.37561049 0.322361 A Allelic ALL hCV25944011 EGLN2 Sample Set 20.036898 1.366324 0.39094994 0.319636 A Allelic Male hCV313778 GOLGA5Sample Set 2 0.022064 0.751072 0.2535017 0.3113598 A Allelic ALLhCV313778 GOLGA5 Sample Set 2 0.002363 0.52557 0.19889103 0.3208284 AAllelic Female hCV313778 GOLGA5 Sample Set 2 0.019467 0.6962560.24191677 0.314285 A Allelic Smoke+ Note *Odds Ratio (OR) notapplicable because sum score is a continuous end point

1. A method for identifying a human who has an altered risk fordeveloping coronary stenosis, comprising detecting a single nuclcotidepolymorphism (SNP) at position 101 of SEQ ID NO: 7605 in a nucleic acidsample from said human, wherein the presence of T at position 101 of SEQID NO: 7605 For one allele indicates said human is at an increased riskof developing coronary stenosis, and the presence of C at position 101of SEQ ID NO: 7605 for both alleles indicates said human is at adecreased risk of developing coronary stenosis.
 2. The method of claim 1in which detection is carried out by a process selected from the groupconsisting of: allele-specific probe hybridization, allele-specificprimer extension, allele-specific amplification, sequencing, 5′ nucleasedigestion, molecular beacon assay, oligonucleotide ligation assay, sizeanalysis, and single-stranded conformation polymorphism.
 3. The methodof claim 1 wherein said human has a history of smoking.
 4. The method ofclaim 1 wherein said human has T at position 101 of SEQ ID NO: 7605 forboth alleles thereby indicating said human is at an increased risk ofdeveloping coronary stenosis.
 5. The method of claim 1 wherein saidhuman has T at position 101 of SEQ ID NO: 7605 for one allele therebyindicating said human is at an increased risk of developing coronarystenosis.
 6. The method of claim 1 wherein said human has C at position101 of SEQ ID NO: 7605 for both alleles thereby indicating said human isat a decreased risk of developing coronary stenosis.
 7. The method ofclaim 1, wherein the detection is carried out by using a set ofdetection reagents comprising the oligonucleotide sequences of SEQ IDNO: 26185, SEQ ID NO: 26186, and SEQ ID NO: 26187.